Amikacin is used in the systemic treatment of serious infections, but also locally for the treatment of skin infections. The aim of this work was to develop and validate a simple procedure for amikacin determination inside the epidermal tissue: this implies a simple method for an efficient drug extraction from the skin and a clean and easy HPLC analysis. Amikacin was extracted from epidermis samples with 500μl of a mixture methanol–water–0.05M NaOH (5:5:2v/v/v) at 60°C for 1h. After filtration, the obtained solution was derivatized (1-fluoro-2,4-dinitrobenzene at 90°C for 10min) and analyzed by HPLC, on a C18 μBondapack 300mm×4.6mm column thermostatted at 45°C. The mobile phase was a mixture of acetonitrile–water–acetic acid (47:53:0.1v/v/v) at a flow rate of 1.5ml/min and the UV detector was set at 365nm. The derivatization and HPLC analysis were validated in the concentration interval 1.64–49.21μg/ml. The linearity resulted very good (R=0.9995); the R.S.D.% varied between 0.20% and 3.89% depending on the concentration and the ER% was included between 5.4 and 0.9. The extraction method used demonstrated to be specific and the recovery resulted about 93%. The extraction, derivatization and HPLC assay has good reproducibility, sensitivity and specificity resulting in a reliable method for biopharmaceutical studies of AK distribution in the epidermis.