A rapid, sensitive and reproducible high performance liquid chromatographic (HPLC) method was developed and validated in plasma for simultaneous quantification of 5-fluorouracil, adriamycin and cyclophosphamide (FAC) which are being prescribed in cancer chemotherapy. Isocratic RP-HPLC system (Agilent technologies USA) with C 18 column (4.6 mm × 250 mm i.d., 5 µ particle size) and a detector UV-VWD was employed. The mobile phase of 0.05 M disodium hydrogen phosphate and acetonitrile (65:35 v/v) containing 0.5 mL/L triethylamine (pH 3.7) was pumped at 0.650 mL/min. UV detection of FAC was set at 266, 254 and 198 nm, respectively. Total run time was 15 min and retention times for 5-fluorouracil, cyclophosphamide and adriamycin were 4.1, 7.7 and 10.9 min, respectively. Parameters of validation (LOD, LOQ, Linearity, accuracy, precision and sensitivity) were established. The method provides specific quantification for FAC with high resolution. A simple, reproducible and inexpensive HPLC method was developed and applied for pharmacokinetic evaluation in breast cancer patients.