The aim of this study was to validate an HPLC-UV method to assess vitamin D status by determining the linearity and precision of the 25-hydroxyvitamin D₃ (25(OH)D₃) calibration curve, the limits of detection, quantitation and robustness of the method, and its accuracy. A second stock solution of 25(OH)D₃ was prepared (500 ng/mL), and working dilutions (5, 10, 20, 30, 40, and 50 ng/mL) were prepared for a calibration curve. The HPLC equipment had a UV-Vis diode-array detector and utilized an Acclaim^TM 120 C18 column (5 µm, 4.6 × 250 mm) with a flow rate of 1.2 mL/min, a column temperature of 30 °C, and the standards and samples were maintained at 4 °C, with an injection volume of 100 µL. Detection of 25(OH)D₃ was determined at 265 nm, with a retention time of 4.0 min. The validation was conducted according to the FDA Validation of Analytical Procedures: Guidance for Industry. Vitamin D was extracted from plasma samples using acetonitrile (ACN)–0.1% formic acid (2:1 v/v), and the percentage of recovery was calculated. The proposed method conditions gave excellent linearity (R² = 0.9989) and the linearity coefficient was R² > 0.99 for 25(OH)D₃. The detection and quantification limits were 1.1703 ng/mL and 3.5462 ng/mL, respectively. Decreasing or increasing the reading temperature by 1 °C decreased the response units (AU) of vitamin D, 25(OH)D₃. When the current flow rate decreased by 0.2 mL/min (1.0 mL/min), the retention time increased to 4.913 min, whereas an increase of 0.2 mL/min of the proposed flow rate (1.4 mL/min) decreased the retention time to 3.500 min. The percentage of recovery varied from 92.2% to 97.1%. The proposed method to quantify a vitamin D metabolite (25(OH)D₃) in human plasma samples was reliable and validated.