A single high-performance liquid chromatography (HPLC) method, suitable for the analysis of daunorubicin, idarubicin, doxorubicin, epirubicin, and their 13-dihydro metabolites is validated in the present study. Preparation of plasma samples was performed by a first extraction of analytes with a chloroform/1-heptanol mixture (9:1) and reextraction with ortophosphoric acid 0.1 M. The chromatographic analysis was carried out by reversed-phase isocratic elution of anthracyclines with a Supelcosil LC-CN 5 mm column (25 cm x 4.6 mm internal diameter; Supelco) and detection was accomplished by spectrofluorimetry at excitation and emission wavelengths of 480 and 560 nm, respectively. All anthracyclines eluted within 15 minutes of injection and the method appeared to be specific, because the chromatographic assay did not show interferences at the retention time of analytes. The linearity, evaluated over a concentration range of 0.4-10,000 ng/mL, gave regression coefficients better than 0.999, with recoveries of doxorubicin-doxorubicinol and epirubicin-epirubicinol of 67%-109% and 61%-109% respectively, and 93%-109% for the other compounds. The limits of detection and quantification were 0.4 ng/mL in a 50-mL sample (40 pg/injection) for all anthracyclines tested. The method proved to be precise and accurate, as the within-day and between-day coefficients of variation were less than 10% and the accuracy of the assay was in the range of 91%-107%. Overall results indicate that it is feasible to analyze all the anthracyclines used in clinical practice and their major metabolites with a single optimized method, thereby simplifying their monitoring in chemotherapeutic regimens of cancer patients.