<p>The goal of the present investigation was to develop a simple, accurate, precise, robust, and cost-effective UV-spectroscopic technique for the quantitative estimation of xanthohumol in bulk and hydrogel, in accordance with the International Conference on Harmonisation guidelines. The λ_max of xanthohumol was found to be 363 nm with <i>y</i> = 0.06363<i>x</i> + 0.0115 as the regression equation, a 0.9968 regression coefficient, and showed linearity in the range 2–14 μg/mL. Relative standard deviation (%RSD) values for intraday analysis were 1.105, 0.936, and 0.32, whereas for interday analysis, these values were 1.312, 0.433, and 0.979 (&lt;2% RSD). RSD for absorbance values analyzed using different spectrophotometers was 0.886 and 1.242%, and by different analysts it was 0.647 and 1.044% (&lt;2% RSD), which validated the ruggedness parameter. RSDs for absorbance values analyzed at 361, 363, and 365 nm were 0.761, 1.045, and 0.885%, respectively (RSD &lt; 2%), indicating robustness. The mean recovery of xanthohumol in the presence of carbopol-934 was in the range 98–102%, which validated its specificity. The limit of detection (LOD), limit of quantitation (LOQ), and Sandell’s index for xanthohumol were 0.639, 1.939 μg/mL, and 0.017278 μg/cm² per 0.001 absorbance unit respectively, indicating higher sensitivity of the analytical method for xanthohumol detection. Xanthohumol-loaded carbopol-934 hydrogel was formulated by dispersing xanthohumol in carbopol-934 and neutralizing it with triethanolamine. The solubility of the bulk form of xanthohumol in phosphate buffer at pH 6.8 was 1.153 μg/mL, which was enhanced to 4.12 μg/mL by the production of xanthohumol-loaded Carbopol hydrogel.</p>