Oral route is preferred for drug administration due to convenience in dose administration, lack of pain, effectiveness and increased patient compliance. Drug’s decreased aqueous solubility, gastric instability, enzymatic degradation, decreased biological membrane permeability, P-gp based drug efflux, first pass metabolism, mucus membrane barrier and early clearance from the body are various factors leading to poor oral bioavailability and less therapeutic efficacy of orally administered drugs. Conventional drug dosage forms have not been effective in addressing these issues. Recently, nanotechnology has got much scientific interests in various fields. The biomedical applications of nano-carriers have been the subject of recent interests due to their evident role in addressing the above mentioned drug related issues. Amphiphilic molecules are capable of self-assembling into vesicles upon their contact with aqueous media. They are able to dissolve both hydrophilic and lipophilic drugs either in their interior aqueous compartment or exterior lipid bilayer respectively. Self-assembling amphiphilic molecules like phospholipids, nonionic surfactants, block copolymers and peptides are widely used for designing vesicular nanocarrier drug delivery systems. They are physically stable, economic, efficient and release the loaded drug in a sustain manner, thus leading to enhance oral bioavailability with better clinical outcomes. The current study was designed for the synthesis of four different biocompatible amphiphiles for nano-vesicular drug delivery applications. They were synthesized in neucleophilic substitution reactions by reacting hydrophilic moieties (Creatinine and Pyridoxine) with lipophilic acyl chlorides and aliphatic alkyl bromide respectively. The Vitamin E based amphiphile was synthesized in multi step reaction to introduce polar hydrophilic moiety in lipophilic alpha tocopherol (Vitamin E). These synthetic amphiphiles were characterized through 1H NMR and xii Mass spectrometric analytical techniques. All the amphiphiles were subjected to biocompatibility studies and their nano-vesicular drug delivery applications were investigated using model poorly aqueous soluble drugs i.e. Clarithromycin, Azithromycin and Amphotericin B. All the amphiphiles were less cytotoxic, hemo-compatible and non-toxic to animals. They were capable of forming nano-vesicles of almost spherical shape with homogenous size distribution. The drug loaded vesicles exhibited increased surface negativity and loaded increased amounts of the selected model drugs. They released the loaded drugs in a controlled pattern at various pHs when tested for in-vitro release. Amphiphiles based drug loaded vesicles demonstrated higher storage stability and retained maximum drugs upon incubation with simulated gastric fluid. Invivo bioavailability of the selected drugs was increased in a controlled manner in rabbits upon their oral administration in the form of amphiphiles based nano-vesicles. Results of the current study authenticates biocompatibility of these synthesized amphiphiles and their effectiveness for oral drug delivery of poor aqueous soluble drugs. The results are interesting and provide a scientific base for their use in vesicular drug delivery systems. The results will open new research areas for formulation scientists to investigate their membrane permeability mechanisms with ultimate increased therapeutic efficacy of loaded drugs.