The scope of this study was the development and validation of a reliable, rapid, simple, sensitive, precise reversed-phase high performance liquid chromatography (RP-HPLC) method for the determination of nateglinide in drug substances. The method was developed on ACE C-18 analytical column (150 x 4.6 mm i.d., particle size 5.0 mu m) using a mobile phase of acetonitrile and 0.05% trifluoroacetic acid (25: 25, v/v). The eluent was monitored with UV detection at 210 nm at a flow rate of 1.5 mL/min. Calibration curve was linear between the concentration range of 0.2846-1.0125 mg/mL. The retention time of Nateglinide was 7.07 min and the correlation coefficient (r) of the regression equation for the Nateglinide was greater than 0.99 in all cases.