People all over the world are affected by the chronic, disabling autoimmune illness known as rheumatoid arthritis (RA). Herbal products are more commonly used in treating RA as of their safety and efficacy. The selected formulation for the present study is ANTARTH capsules, which is a polyherbal formulation containing different herbs effective in the treatment of RA. The purpose of this study was to develop and validate a precise, accurate, novel high-performance thin-layer chromatography (HPTLC) method for the simultaneous estimation of quercetin, berberine, rutin, and curcumin and to determine the levels in a polyherbal formulation. A good chromatographic separation was accomplished using a mobile phase consisting of toluene‒ethyl acetate‒methanol‒formic acid (5:3:2:0.5, V/V ) with wavelengths of 366 nm and 426 nm using an ultraviolet‒visible (UV‒VIS) detector. The retention factors for quercetin, berberine, rutin, and curcumin were found to be 0.57, 0.316, 0.093, and 0.663, respectively. A good linear regression relationship between peak areas and concentrations was obtained with correlation coefficients of 0.9988, 0.9987, 0.9977, and 0.9984 for quercetin, rutin, berberine, and curcumin, respectively. The method developed was found to be accurate, precise, and robust. The method was then applied in a marketed formulation (ANTARTH capsule), and the percent content was found to be 5.26 ± 1.09, 4.76 ± 0.83, 3.80 ± 0.94 and 7.62 ± 1.58 for quercetin, rutin, berberine, and curcumin, respectively.