A rapid, specific, and reliable technique has been developed for the determination of lubiprostone in surface of pharmaceutical equipment’s by high performance liquid chromatography. For that study, reverse phase liquid chromatography was employed, using 0.01 M Potassium dihydrogen orthophosphate buffer, pH=3.0 and Acetonitrile (50:50 v/v) and a detector wavelength of 205 nm. The instrument was set at a flow rate of 1.0 mL min−1 and column at ambient temperature. The method showed excellent linearity over a range of 0.5–30 µg mL−1. The swabbing procedure optimized to achieve suitable accuracy in stainless still plate. The accuracy was found >90%. This method is suitable for trace level quantification of Lubiprostone in production equipment surface area and confirm the cleaning validation of the production area and equipment’s for avoiding the cross contamination of Lubiprostone to the other products.