A modified high performance liquid chromatography (HPLC) method for the quantification of vancomycin levels in plasma and tissues is described. The method uses solid phase extraction (SPE) of vancomycin from the samples and reversed phase HPLC with UV detection. The method was fully validated in terms of recovery, linearity, selectivity and various stability conditions. Vancomycin was determined in plasma samples obtained from 15 patients undergoing cardiopulmonary bypass, before and repeatedly during 12 h after drug administration. The vancomycin levels in plasma were measured by HPLC and by fluorescence polarization immunoassay (FPIA) (TDX). The following correlation was found: TDX = 0.84 HPLC + 1.04. The mean vancomycin levels in skin, fat, atrium, pericardium and sternum, before and after bypass, are reported.