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Background: 5,7-Dihydroxyflavone (Chrysin) is a naturally occurring flavonoid with diverse pharmacological activities, necessitating reliable analytical methods for its quantitative estimation. This study aimed to develop and validate a simple, rapid, and cost-effective UV spectrophotometric method for the determination of 5,7-Dihydroxyflavone. Methods: The method was developed using methanol as the solvent, and the absorption spectrum was recorded over the wavelength range of 200–400 nm. Quantification was performed at the maximum absorption wavelength (λmax) of 313 nm. Calibration standards were prepared in the concentration range of 4–20 μg/mL. The proposed method was validated according to the International Council for Harmonisation (ICH) Q2(R2) guideline for specificity, linearity, accuracy, precision, robustness, ruggedness, limit of detection (LOD), limit of quantification (LOQ), and solution stability. Results: The method exhibited excellent linearity over the concentration range of 4–20 μg/mL with a correlation coefficient (r²) of 0.9992. The LOD and LOQ were found to be 1.37 μg/mL and 3.29 μg/mL, respectively, indicating satisfactory sensitivity. Accuracy studies demonstrated recoveries within acceptable limits, while precision, robustness, and ruggedness showed %RSD values below 2%, confirming the reproducibility and reliability of the method. No interference from the solvent was observed at the selected analytical wavelength. Conclusion: The validated UV spectrophotometric method is simple, accurate, precise, sensitive, and economical, making it suitable for the routine quantitative estimation and quality control of 5,7-Dihydroxyflavone in pharmaceutical and research laboratories.