A new stability-indicating HPTLC assay has been established for leuprolide acetate. Silica gel 60F 254 was used as stationary phase. Samples and reference standards were applied to the same precleaned and activated HPTLC plates and chromatography was performed in an automated multiple development chamber (AMD2) with five-step isocratic incremental multiple development with 1 min drying time between each step and layer conditioning with the mobile phase before each development. The mobile phase, ethyl acetate-methanol-25% aqueous ammonia, 60 + 30 + 10, resulted in dense, compact zones for the analyte and related substances. Leuprolide acetate was subjected to acidic, basic, and oxidative degradation. The peaks of the degradation products were well resolved from the main peak with significantly different migration distances. Densitometric evaluation was performed at λ = 280 nm. The calibration function of the analyte was linear in the range 107–422 ng and the correlation coefficient was 0.9914. The limits o...