A simple, rapid, and sensitive high-performance liquid chromatography method was developed and validated for the simultaneous determination and quantification of fusidic acid and steroids (prednisone, betamethasone valerate, hydrocortisone acetate, and dexamethasone sodium) from bulk drugs and human plasma. A RP-HPLC, operated at ambient temperature, was equipped with a UV detector for monitoring the effluents at 235 nm. The mobile phase consisted of methanol, acetonitrile, and 0.05 M phosphoric acid (10:60:30, v/v/v), and separation was achieved on a Medeterrane, C18 (5 μm, 12.5 × 0.46 mm) column at a flow rate of 1.7 mL min -1. Calibration curves were linear over concentration range 0.625–10 μg mL -1 with correlation coefficient (r2) greater than 0.9999. The coefficient of variation (CV) and relative error (RE) for intra- and interassay were <2% and <1%, respectively. Interference of other already administered common medicaments, such as aspirin, paracetamol, caffeine, nicotine, and other plasma components, were not found.