An HPLC method for Vitamins A and E in rat plasma has been developed. The main goals of the method are the small amount of sample, 50 microl, and the direct extraction of analytes in one step with acetone, which is a solvent compatible with the reverse-phase mobile phases. Recoveries, as compared with classical and more tedious methods, were near 100%. The method employs a Supelco Discovery C18 column and methanol/water (95:5, v/v) as mobile phase. After being developed, the method was validated following ICH guidelines, with UV, fluorescence and electrochemical detectors. It proved to be selective, lineal, accurate and precise. This method greatly simplifies sample treatment and that is a critical point when working with a large number of samples.