A simple, sensitive and selective high performance liquid chromatographic method with UV detection for the chiral separation of racemic methotrexate (rac-Mtx) and enantiomeric purity of L-methotrexate in pharmaceutical formulations was developed and validated. The chiral separation was optimized studying both the nature of the stationary phase by using Chirobiotic T, Chiracel OJ and human serum albumin columns and the effect of the mobile phase composition. The best results in terms of enantioresolution and enantioselectivity were achieved with a polar organic mobile phase on Chirobiotic T stationary phase. Essential steps in method validation such as precision, accuracy, suitability and stability were studied according to ICH guidelines. At wavelength 303 nm, the limit of detection (S/N=3) was found to be 0.9 microg/ml for rac-Mtx. The separation of D-Mtx at 0.2% (w/w) level (as limit of quantitation) from the main drug L-Mtx was successfully obtained with 1.72 enantioresolution value. Enantiomeric purity of L-Mtx was determined in pharmaceutical formulations (tablets and injections) with inter- and intra-days relative standard deviation < or = 1.6%. Under the validated stereoselective HPLC conditions for methotrexate, folic acid was also analysed.