Abstract: To identify the related substances in pioglitazone hydrochloride by hyphenated LC-MS techniques, an Ultimate XB-C₁₈(250 mm×4. 6 mm, 5 μm)column was used for separation of the related substances with methanol and 0. 1% ammonium acetate buffer as the mobile phases in gradient elution. Electrospray positive ionization high resolution TOF/MS was used for the determination of the accurate mass and elemental composition of parent［M+H］⁺ ions of the related substances, and triple quadrupole tandem mass spectrometry was employed for the product mass spectra determination. Eleven major related substances were detected and identified to be one synthesis intermediate, six by-products and four degradation products, by using LC-MS determination, spectra elucidation, and further synthetic process and stress degradation mechanisms analysis. The results are useful for pioglitazone hydrochloride manufacturing processes optimization and quality control.