The natural methyl donor group, S-adenosylmethionine and its product, S-adenosylhomocysteine play an important role in many biochemical reactions involving transmethylation reactions. These compounds can be used as biomarkers in incipient diagnosis of various pathological disorders therefore the validation of a suitable method to routinely analysis of these compounds is very important. In this paper, a high performance liquid chromatrography method for S-adenosylmethionine and S-adenosylhomocysteine measurement as fluorescent 1,N(6)-ethanoderivatives from biological samples was validated in terms of selectivity, linearity range of the response (R > 0.9993), detection limit (9 × 10(-9) and 4.4 × 10(-9) mol L(-1)), the limit of quantitation (9.7 × 10(-9) and 5.7 × 10(-9) mol L(-1)), precision, trueness and robustness. The method for quantification simultaneous of these compounds is rapid, sensitive and precise and appropriate for clinical analysis.