A novel analytical method, based on high-performance liquid chromatography with a UV (HPLC-UV) detection system for the sensitive detection of a genotoxic impurity (GTI) 5-amino-2-chloropyridine (5A2Cl) in a model active pharmaceutical ingredient (API) tenoxicam (TNX), has been developed and validated. The HPLC-UV method was used for the determination of GTI 5A2Cl in API TNX. The compounds were separated using a mobile phase composed of water (pH 3 adjusted with orthophosphoric acid): MeOH, (50:50: v/v) on a C-18 column (150 x 4.6 mm i.d., 2.7 mu m) at a flow rate of 0.7 mL min(-1). Detection was carried out in the 254 nm wavelength. Column temperature was maintained at 40 degrees C during the analyses and 10 pL volume was injected into the HPLC-UV system. The method was validated in the range of 1-40 mu g mL(-1). The obtained calibration curves for the GTI compound was found linear with equation, y = 40766x - 1125,6 (R-2 = 0.999). The developed analytical method toward the target compounds was accurate, and the achieved limit of detection and limit of quantification values for the target compound 5A2Cl were 0.015 and 0.048 mu g mL(-1), respectively. The recovery values were calculated and found to be between 98.80 and 100.03%. The developed RP-HPLC-UV analytical method in this research is accurate, precise, rapid, simple and appropriate for the sensitive analysis of target GTI 5A2Cl in model API TNX.