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A selective and rapid reversed-phase HPLC-UV method was developed and validated to quantify tavaborole (TAV; AN2690) in biological samples, i.e., in receiving phase and in bovine hoof membrane extract derived from in vitro transungual permeation studies. A simple solid-liquid extraction procedure was used to recover the drug from the bovine hoof slices. TAV chromatographic separation was achieved on a Luna PFP column (150 × 4.6 mm, 5 μm) using a mobile phase consisting of a 70% phosphoric acid solution (10 mM, pH 2.0) with 30% acetonitrile. The detection wavelength was set to 220 nm using a UV detector. The method exhibited good linearity in the calibration ranges, which were 0.5-8.0 and 0.03-2.5 μg/mL for the receiving phase and hoof membranes, respectively. The obtained LOD and LOQ values were 0.023 and 0.069 μg/mL, respectively, for the receiving phase and 0.0024 and 0.007 μg/mL for the bovine hoof membrane extracts. In all cases, the CV for intraday and interday precision was widely below the limit of 2%, demonstrating good precision. The analytical method described was sensitive, precise, linear, and accurate and could be applicable for clinical and bioanalytical studies as an alternative to other analytical methods, which are quite expensive and not always available in research laboratories.