Background: Propylthiohinothiadiazole is a thiadiazole derivative with pronounced antimycobacterial action. Substance is researched in order to develop methods of analysis and standardization, analytical documents regulating quality for use as an active principle in the development of pharmaceutical forms. Material and methods: 3 series of propylthiohinothiadiazole, internal reference standard of 2-(propylthio)-5H-[1,3,4]-thiadiazole[2,3-b]-quinazolin5-one substance with concentration 99.98%, chromatographic system Shimadzu LC-20AD high-pressure liquid chromatography (HPLC) with UV-VIS detector, acetonitrile and dimethylsulfoxide of purity grade “pro HPLC analysis” (Sigma Aldrih and Merck), bidistilled purified water. Results: Linearity is proved for concentrations of 5-30 μg/ml, the linear regression equation is y=36472x-44580, R²=0.9999. The limit of detection is 0.729 μg/ml and the limit of quantification is 2.210 μg/ml. It was established that the method is accurate (mean recovery values at 80%, 100% and 120% concentration levels were close to 100%). The accuracy of the method was expressed by repeatability and intermediate accuracy. The variation of the chromatographic conditions established that the method is robust. For all validation parameters, relative standard deviation was less than 1. Conclusions: The validation results show that the developed HPLC method is simple, fast, accurate and reproducible.