The measurement of endogenous estradiol and estriol levels in the skin is crucial for understanding their physiological roles and therapeutic applications. In vitro permeation testing (IVPT) is a key method for studying substance diffusion through the skin, but it faces challenges such as maintaining sink conditions and intra-subject variability. The purpose of this study is to evaluate the permeation and retention of endogenous estradiol and estriol in human skin using IVPT and extraction methods. The results from this research will help to facilitate the development of personalized dermatological treatments tailored to individual skin characteristics, enhance the effectiveness of transdermal drug delivery systems, and inform the formulation of cosmetic products designed for anti-aging and skin hydration.