Commelina benghalensis (Commelinaceae) is widely used as traditional and folklore medicine in India. In the present study, a reverse-phase high-performance liquid chromatography—photodiode array detection (RP-HPLC—PDA) method was developed for the separation, identification, and quantification of bioactive phenolics. Antioxidant potential was also accessed to validate the presence of identified markers. Method was developed on C18 column with 1% formic acid (in water) and acetonitrile as solvent system, and data acquisitions were achieved at wavelength of 285 nm. The developed method was also validated for accuracy, precision, robustness, limit of detection and quantification (LOD and LOQ), repeatability, and recovery according to International Conference on Harmonization (ICH) guidelines. In this method, five phenolics, viz., protocatechuic acid (0.033%), vanillic acid (0.262%), ferulic acid (0.365%), apigenin (0.126%), and kaempferol (0.544%), were quantified in linearity range of 0.2–1.0 μg with correlation coefficient of more than 0.9949. Relative standard deviation (RSD) (%), LOD, LOQ, and recovery (%) are within the acceptable limit. Besides that, methanolic extract shows the inhibition (%) range from 24.45 to 68.75% at 0.02–0.12 mg Ml-1. IC50 of extract was observed at 46.75 μg Ml-1, suggesting the promising activity in methanol extract. Hence, the proposed method for simultaneous quantification of five bioactive phenolics in the tuber of C. benghalensis using HPLC–PDA detection under the specified conditions is specific and accurate, and validation proves its selectivity and reproducibility.