The aim of the present study was to modify and validate a high-performance liquid chromatographic (HPLC) method for determining 2,3 and 2,5 dihydroxybenzoic acid (2,3-DHBA and 2,5-DHBA) from salicylic acid in human plasma. The mobile phase was a mixture of sodium acetate/citrate (pH 2.5) 30 mM-methanol (93:7, v/v). The injection volume was 10 muL. Retention time for 2,5-DHBA, and 2,3-DHBA was 4.5 +/- 0.10 and 5.8 +/- 0.15 min, respectively. The detection and quantification limits were 10 and 40 nM for 2,3-DHBA and 8 and 20 nM for 2,5-DHBA. Linearity was evaluated in the range of 40-1600 nM for both metabolites. Inter- and intra-analysis variation coefficient was below 10%. Good recoveries of more than 99% were obtained for both metabolites using this method.