This study investigates the medicinal potential of Justicia Adhatoda Vasica (JAV) by focusing on the isolation, phytochemical analysis, and in-vivo analgesic activity of its bioactive compound. Then obtained crude extract underwent isolation process. The in-vivo analgesic activity of the compound was assessed, shedding light on their potential in pain relieving effect. In the Preliminary TLC of JAV Methanolic extract was performed and Rf values were found to be 0.51 and 0.51 of JAV and Std. Flavonoid. Advanced spectroscopic techniques like UV spectroscopy, FT-IR spectroscopy, Mass spectroscopy, NMR were employed for phytochemical analysis to identify and characterize the isolated compound. The study recorded UV spectra of isolated fraction of JAV, determining λmax and wavelength of 292 nm. Physical, chemical, and spectral investigation confirmed the presence of 5,7-dihydroxy-2-(4-hydroxyphenyl)-2,3-dihydrochromen-4-one in JAV Methanolic extract fraction (J). In-vivo analgesic study found that oral administration of both extract and isolated compound at 200 and 40 mg/kg and standard drug significantly decreased the number of licking induced by formalin compared to the control group, with significant effects observed at all experimental doses. Hence, this research provides valuable insights into the pharmacological properties of Justicia Adhatoda Vasica, laying the groundwork for further exploration of its therapeutic applications