The present study aimed to evaluate the antioxidant activity and perform high-performance thin-layer chromatography (HPTLC) analysis of the ethyl acetate fraction of the acetone extract of Hardwickia binata Roxb. The ethyl acetate fraction was assessed for in-vitro antioxidant activity using various methods, including the Cupric Reducing Antioxidant Capacity (CUPRAC) assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay, hydroxyl free radical scavenging assay, reactive nitrogen oxide scavenging assay, superoxide anion radical scavenging assay, ABTS radical scavenging assay, and Ferric Reducing Antioxidant Power (FRAP) assay. The half-maximal inhibitory concentration (IC₅₀) values were determined for each assay: CUPRAC (4.9 ± 1.1 µg/mL), DPPH (39.05 ± 0.09 µg/mL), hydroxyl free radical scavenging (11.62 ± 0.0931 µg/mL), reactive nitrogen oxide scavenging (435.2 ± 0.01 µg/mL), superoxide anion radical scavenging (16.89 ± 0.06 µg/mL), ABTS radical scavenging (14.9 ± 1.1 µg/mL), and FRAP (2.442 ± 0.12 µg/mL).. HPTL Among these, the CUPRAC and FRAP assays exhibited the most significant antioxidant activity C fingerprinting of the ethyl acetate fraction under UV light revealing the presence of seven components with retention factor (Rf) values of 0.012, 0.073, 0.12, 0.18, 0.569, 0.658, and 0.935. The ethyl acetate fraction was further analyzed for quercetin content using HPTLC densitometric analysis with a solvent system comprising toluene: ethyl acetate: methanol: glacial acetic acid (5:4:0.5:0.5, v/v). The analysis was performed on precoated silica gel 60F254 plates, and densitometric detection of quercetin was conducted at 254 nm and 366 nm. The amount of quercetin was quantified by comparing with a standard calibration curve (linear range: 200–600 ng/spot, r² = 0.9989). The concentration of quercetin in the ethyl acetate fraction was 43.41 µg/mg of the dry fraction. The results indicate that Hardwickia binata contains natural antioxidants.