Modern farming practices involve administration of a wide range of veterinary drugs and biological substances to food-producing animals with the primary aim to combat diseases and promote growth. The residues of these drugs can enter into food chain either directly or indirectly resulting in serious allergic reactions in humans, and development of drug resistance in multiple strains of bacteria as in case of antibiotics. The main focus of this research work was the development of analytical methodologies for the identification of various classes of antibiotics in broiler chicken and bovine milk using LC-MS/MS. All the developed methods were validated according to European Commission Decision 657/2002/EC and parameters determined are: specificity, accuracy, precision, repeatability, within lab reproducibility, decision limits (CCα) and detection capability (CCβ). The first method developed for the analysis of florfenicol residues in chicken muscles. It was found that most of the florfenicol residues remained bound with tissues and could only be extracted with organic solvents after digestion of the tissues with strong hydrochloric acid at 95-100 °C. The bound residues of florfenicol were present in higher concentration as compared to un-bound residues in various tissues of broiler chicken. The second method was developed for the multi-amphenicol analysis in which florfenicol, florfenicol amine, chloramphenicol and thiamphenicol were simultaneously analyzed. The accuracy values were 86.4 to 108.1% and precision values were 2.7 to 16.3%, respectively. Analysis of the various raw samples confirmed the presence of florfenicol and chloramphenicol residues in few samples at low levels. Third method involved multiclass multidrug residues, in which total 68 compounds from six antibiotic classes were analyzed. The accuracy values were 70- 102.9 % for nearly 87% compounds. The developed method can be used for screening purpose to reduce the number of samples for confirmatory analysis, thus improving the efficiency of the laboratory by reducing cost and time. Lastly, prevalence of penicillin and cephalosporin antibiotics was studied in bovine milk samples obtained from two cities of Punjab, Pakistan using UPLC-MS. Only amoxicillin, ampicillin, Pen G and cloxacillin were found and confirmed in 18 samples.