Spodoptera litura (Fabricius) is an economically important pest of many agricultural crops, to study the effects of acetamiprid, acephate, fipronil and custard apple Annona squamosa L. on cotton pest Spodoptera litura (Fab.), with reference to transgenic and conventional cotton varieties studies were carried out. Five doses were used for each under test samples i.e., acetamiprid 0.16, 0.20, 0.24, 0.28, 0.32 %, acephate 0.097, 0.291, 0.485, 0.679, 0.873 %, fipronil 0.01, 0.02, 0.03, 0.04, 0.05 %, and Annona squamosa 0.9, 1.1, 1.3, 1.5, 1.7 % concentrations of a desired volume were prepared by diluting in distilled water. Sixty larvae were treated by using leaf dipping method. After 72 hours mortality was recorded. The experiment was replicated four times. By calculating the mean mortalities through probit analysis in respect to various doses of the insecticides and custard apple Annona squamosa LC50 was calculated, as the pattern of toxicity showed, fipronil was found to be the highest toxic with the lowest LC50 value of 0.025% followed by acetamiprid 0.231%, acephate 0.337% and Annona squamosa with 1.319% respectively. Same numbers of Spodoptera litura larvae were treated with LC50 of each insecticide and Annona squamosa, and the fresh leaves of 40 days aged cotton plant of respective variety to study their effects on biotic potential of Spodoptera litura. Acetamiprid found to be highly effective against pupation. Fipronil and Annona squamosa was found to be the more promising with pupal weigh of 292.75 and 300.21 mg/pupa respectively. Annona squamosa and cotton varieties caused prolong pupal duration as compared to other treatments whereas the lowest pupal duration was recorded in fipronil treatment. Acephate showed higher toxic effects against adult emergence followed by acetamiprid, fipronil, Annona squamosa, CEMB-33, Bt-906, FH-142 and CIM-496. The lowest adult female/male ratio was observed in fipronil, acephate and CEMB-33 treatment as 0.866, 0.867 and 0.936, female/male while it was found almost equal in Annona squamosa and it was higher in cotton varieties. To determine the effects on biotic potential of Spodoptera litura four different mating combinations of male and female crosses were established for adults emerged from each concentration treated larvae i.e., T♀xU♂, T♂xU♀, T♀xT♂ and U♂xU♀ as control. Adult when crossed as T♀xU♂, Annona squamosa and insecticides were found to be more effective than under test cotton varieties against fecundity as well as fertility. Among insecticides and Annona squamosa, higher effects were recorded in acephate followed by Annona squamosa, fipronil and acetamiprid. Among cotton varieties CEMB- 33 found to be the most effective followed by Bt-906, FH-142 and CIM-496 as compared to control. Adults when crossed as T♂xU♀, acephate and Annona squamosa, showed higher effects in insecticides and in cotton varieties CEMB-33 were found to be the most effective against fecundity and Annona squamosa showed higher effects on fertility followed by fipronil, acephate and acetamiprid and in cotton varieties CEMB-33 showed higher effect against fertility of Spodoptera litura. while in the case of T♀xT♂ fecundity was found tremendously suffered, acephate and Annona squamosa was found to be effective than others, where as fipronil, acetamiprid and Annona squamosa proved to be the most effective against fertility. Adults, pairs in all crosses acephate treated adults showed the lowest incubation period whereas Bt-906 and CEMB-33 showed higher incubation period than in control. Newly emerged T♀xU♂, Annona squamosa showed the lowest adult male longevity, whereas in the same pair acephate showed the lowest female adult. When adults were paired as U♀×T♂ of Spodoptera litura, acephate showed the lowest adult male longevity, whereas fipronil and Annona squamosa showed the lowest female adult longevity. In the case of T♀xT♂ acephate and Annona squamosa showed the lowest adult male and female longevity. Sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was employed to determine the effects of insecticides, Annona squamosa, and under test cotton varieties on the protein pattern of 5th instar Spodoptera litura larvae. Relative flow values and movement of separated proteins were recorded and compared with the protein pattern of untreated group of larvae. Estimation of electrophoretic mobility of different proteins, total protein contents, acetylcholinesterase activity and total esterase activity were determined under the toxic effect insecticides, Annona squamosa, and under test cotton varieties. Quantitative changes in total protein contents and inhibition in the activity of acetylcholinesterase and fluctuation in total esterases were observed. Annona squamosa showed significant toxic effects against Spodoptera litura larvae and exhibited inhibitory activity against total protein, acetylcholinesterase, and detoxification enzymes total esterase. Annona squamosa and transgenic cotton varieties may be considered as ecosystem friendly approach and could be used as pest control agents in order to reduce use of synthetic pesticides.