The basis of the medical system is medicinal herbs. Herbal combinations for therapeutic alignment often include botanical components. This work aims to make an anti-inflammatory ointment from the Ficus religiosa root bark and test the medicine using a novel HPTLC technique. The flavonoid component from the hydroalcoholic extract was separated by column chromatography. The mobile phase used was a mixture of toluene, ethyl acetate, and methanol in the ratio 04:04:02. By using the fusion method to create a straightforward ointment from this separation, it was discovered that the formulation’s physicochemical properties were all within acceptable bounds. The mobile phase for the HPTLC study included toluene, ethyl acetate, formic acid, and methanol (3:3:0.8:0.2), and the wavelength was 280 nm. Using albino mice and the Carrageenan-induced paw edema method, the anti-inflammatory effect was assessed. Rf value is discovered to be 0.86, 0.88, and 0.89 for simple ointment, fractionate, and standard quercetin, respectively. According to ICH criteria, the HPTLC method was validated, and all parameters were within acceptable ranges. The created ointment can be used in a straightforward manner to alleviate inflammation. In comparison to NSAIDS, the topical formulation showed good anti-inflammatory action in carrageenan-induced paw edema.