Genistein (GEN) has potential advantages for topical skin delivery, but no literature data are available for its quantitation in different skin layers, such as the stratum corneum (SC). Therefore, a simple, rapid, selective and sensitive bioanalytical method was developed and validated for GEN quantitation in porcine skin samples following in vitro permeation studies. GEN was assayed by HPLC with UV-Vis detection (270 nm) using 0.5% acetic acid in water–n-propanol–acetonitrile (50 : 2 : 48, v/v/v) as mobile phase (flow-rate of 1.0 mL/min). Specificity was demonstrated since endogenous skin components did not interfere with GEN peak. Standard analytical curve was linear over the concentration range (0.1–60 µg/mL) and the lower limit of quantitation was determined for different skin layers (100 ng/mL). GEN recovery from skin layers ranged from 95.57 to 97.57%. Permeation studies were carried out using an automated vertical diffusion cell apparatus. No fluctuation on the amount of GEN retained in the SC was observed over time, but increasing amounts of the drug were found in deeper layers of the skin. The method was reliable and reproducible for the quantitation GEN in skin samples enabling the determination of the cutaneous penetration profile of this drug in permeation experiments.