OBJECTIVE The purpose of this study was to demonstrate the use of near-infrared (NIR) spectrometry for the in vitro quantification of econazole nitrate (EN) and estradiol (EST) in human skin. METHODS NIR spectra were collected from EN and EST powders to verify the presence of NIR chromophores. One percent EN cream, a saturated solution of EN, or 0.25% EST solution was applied to human skin. NIR spectra were collected and one-point net analyte signal (NAS) multivariate calibration was used to predict the drug concentrations. NIR results were validated against known skin concentrations measured by high-pressure liquid chromatography (HPLC) analysis of solvent extracts. RESULTS NIR spectroscopy measured dermal absorption from saturated solutions of EN on human skin with an r2=0.990, standard error of estimation (SEE)=2.46%, and a standard error of performance (SEP)=3.55%, EN cream on skin with an r2=0.987, SEE=2.30%, and SEP=2.66%, and 0.25% solutions of EST on skin with an r2=0.987, SEE=3.30%, and SEP=5.66%. Despite low permeation amounts of both drugs through the stratum corneum into human tissue, the NIR signal-to-noise ratio was greater than three, even for the lowest concentrations. CONCLUSIONS NIR analyses paralleled the results obtained from HPLC, and thus could serve as a viable alternative for measuring the topical bioavailability/bioequivalence of different EN and EST formulations. Because these experiments were conducted in human tissue, this research suggests an all-optical in vivo method of measurement for dermal absorption could be developed.