Thiowurtzine, a hexaazaisowurtzitane derivate, is a novel original non-opioid analgesic. In this article, an HPLC–UV method has been developed for rapid detection and quantification of API and related substances contained in thiowurtzine capsules. Six thiowurtzine impurities were separated successfully from each other by isocratic elution using a Luna 5u PFP (2) (150 mm × 4.6 mm, 5 µm) column with 30 µL injection volumes at a wavelength of 254 nm. Commercially unavailable impurities were formed by degradation of thiowurtzine and preliminarily identified by high-resolution LC–MS. This method proved to be robust, linear, repeatable, sensitive, selective and handy.