A direct enantio-, diastereo-, and chemo-selective high-performance liquid chromatographic method was developed for determining the content, enantiomeric purity, and related substances of the chiral antidepressant drug sertraline HCl in a single chromatographic run. The separation was achieved on a chiral stationary phase based on amylose tris(3-chloro-5-methylphenylcarbamate) under reversed-phase conditions. The method was optimized by evaluating the influence of the temperature and mobile phase composition on the retention and selectivity. The application of the single-run approach allowed to baseline resolve all investigated species in less than 15 min, without using buffers or tandem-coupled columns. The chromatographic method was validated according to the guidelines of the Official Medi-cines Control Laboratory and applied to control the content of sertraline HCl and related chiral sub-stances in a generic antidepressant formulation.