The study aims to characterize protransfersome gel as a transdermal drug delivery system for Repaglinide to ensure effective and sustained drug delivery. Repaglinide protransfersomal gel was created using the coacervation phase separation method and studied for vesicular morphology, dimensions, entrapment efficiency, and drug penetration through rat dermis. Numerous edge activator and formulation characteristics employed in the development of the optimal formula. All protransfersomal formulations were described, revealing that the created gel's color and physical state were discernible to the naked eye. The mean particle size of protransfersome ranged from 264 to 341 nm. TEM micrographs of the transfersome suspension revealed that the vesicles possess a homogeneous spherical morphology with a smooth surface. The formulation demonstrated excellent stability at 4 ± 1°C, and after three months of storage, there were no alterations in its liquid crystalline properties, drug content, or other distinctive criteria. The protransfersomal formulation is non-irritating. Consequently, the formulated preparation is deemed safe for topical use. The in vivo pharmacodynamic analysis of protransfersomal gel demonstrated a substantial enhancement in the bioavailability of repaglinide compared to oral drug administration.