This study aimed to develop and validate a high-performance liquid chromatography (HPLC) method for the quantitative analysis of maysin, a major bioactive flavonoid in Eremochloa ophiuroides extract. The analytical method was validated in accordance with the guidelines of the Ministry of Food and Drug Safety and AOAC with respect to specificity, linearity, accuracy, precision, and sensitivity. Specificity was confirmed by comparing the retention times and UV spectra of maysin in standard and extract samples, demonstrating identical retention times and no interference from coexisting compounds at the maysin peak. Excellent linearity was achieved over a concentration range of 2.5–50 mg/L, with correlation coefficients (R²) of 0.9999 for all three calibration curves. The mean regression equation was determined as y = 3687.8x − 1434.8. Accuracy, evaluated using the standard addition method, showed recoveries ranging from 99.2% to 101.7%, with relative standard deviations (RSDs) of 0.2–1.0%, satisfying AOAC acceptance criteria. Precision was assessed at three concentration levels (50%, 100%, and 150%). Intra-day precision results obtained by two analysts over five consecutive days showed RSD values ranging from 0.10% to 1.43%. Inter-day and inter-analyst precision results demonstrated RSD values ranging from 0.92% to 1.50%, meeting the AOAC acceptance criterion for reproducibility (RSD ≤ 4.0%). The limit of detection (LOD) and limit of quantitation (LOQ) were calculated as 1.28 μg/mL and 3.89 μg/mL, respectively. Using the validated method, the maysin content of E. ophiuroides extract was consistently determined to be approximately 11.60–12.30 mg/g across different sample loading levels (50%, 100%, and 150%) evaluated during precision validation. Collectively, these results demonstrate that the validated HPLC method provides sufficient specificity, linearity, accuracy, precision, and sensitivity, and is suitable for the reliable quantitative analysis and quality control of maysin in E. ophiuroides extracts.