A simple, sensitive and reproducible method was developed using UV spectrophotometer for the determination of Misoprostol in a drug substance and pharmaceutical dosage form. It is applicable for the quantification of and assay of drug substances. The study was carried out at 275 nm. The developed method was validated for linearity, accuracy, precision, Limit of Detection (LOD) and Limit of Quantification (LOQ) as per International Conference on Harmonization (ICH) guidelines. Misoprostol was subjected to different experimental stress conditions of oxidative acid, base, hydrolytic, thermal and photolytic degradation. It was found to be stable in oxidative acid, oxidative base and hydrolytic conditions. The method developed was validated as per ICH guidelines and can be used in both bulk and pharmaceutical dosage forms.