This article describes an HPTLC method for the determination of dicyclomine HCL from Injection dosage form. The method employes a precoated silica gel 60 F254 (0.2 mm thickness) on aluminium sheets and a mobile phase toluene: acetone: methanol: conc. ammonia in the ratio of 5.0: 2.0: 1.0: 0.02 (v/v/v/v), having chamber saturation for 30 min at room temperature. The mobile phase was run upto 8cm. The dipping agent sprayed on plate for determination of dicyclomine hydrochloride and plate scanned at 523nm. The Rf value was found to be 0.71 for dicyclomine HCL. The linear detector response was observed between 800 ng.spot-1 to 4000 ng.spot-1 for dicyclomine HCL. The method so developed was validated for its accuracy and precision. The LOD and LOQ were found to be 250 ng.spot-1 and 800 ng.spot-1for dicyclomine HCL. The recovery was carried out by standard addition method. The average recovery was found to be 99.45 % for dicyclomine HCL. Statistical analysis showed that the method was repeatable and selective for the quantitation of the drug in injection dosage form and for routine quality control of raw materials of the drug.