The present article focuses on the quantitative analysis of a polyherbal formulation containing arjuna, guggul, and pushkarmoola in a combined dosage form which is used for its cardioprotective, anti-anginal, and hypolipidemic activities. Quantification of guggulsterones E and Z was done by developing and validating a high-performance thin-layer chromatography method after extraction by methanol. Densitometric evaluation was carried out on precoated silica gel G60 F254 high-performance thin-layer chromatography (HPTLC) plates using toluene—ethyl acetate (8:2, v/v) as the mobile phase. Based on the HPTLC results, the methanol extract of guggulsterone was chosen as it showed a better separation of spots as compared to another extract. After postderivatization of plates with anisaldehyde—sulfuric acid (10%) reagent, the methanol extracts gave different color spots (the arjuna extract gave a blue spot, the guggul extract gave orange and brown spot, and pushkarmoola gave dark blue spot). Scanning and densitome...