The aim of the present work was to develop a high-performance thin-layer chromatography (HPTLC) densitometric analysis technique using the solvent system ethyl acetate:methanol:formic acid:water (20:3:1:2, v/v), which has been developed and validated for simultaneous quantification of four marker compounds (quercetin, rutin, luteolin and vitexin) in Asparagus racemosus, Withania somnifera, Vitex negundo, Plumbago zylenica, Butea monosperma, and Tephrosia purpurea plant extracts. The analysis was performed on HPTLC precoated silica gel 60F254 plates with as the mobile phase (distance of 6.5 cm). Densitometric detections of flavonoids were performed at 254 and 366 nm respectively. The amount of these marker compounds was calculated using the regression equations of the calibration curves, which were linear within a range of 0.40–0.90 µg/spot (r2 > 0.99). The system was found to provide compact spots for quercetin, rutin, luteolin and vitexin (Rf values of 0.96, 0.53, 0.96 and 0.78, respectively). The amounts of these marker compounds present was measured by the method developed and expressed in mg ml–1 of dry extracts of plants. This simple, precise and accurate method gave good resolution from other constituents present in the plant extract and has been successfully applied in the analysis and routine quality control of herbal materials and formulations containing these studied plants.