In the world, the use of herbal medicines has a long recorded history and they have been used for various therapeutic purposes. The polyherbal formulation is a core concept of Ayurvedic medicine that prevents unnecessary suffering from disease and promotes long healthy life. The objective of the present study was to do quantitative phytochemicals determination of Drakshavaleha. Drakshavaleha is an Ayurvedic polyherbal formulation prescribed for Pandu (Anemia) and Kamala (Jaundice). High-performance thin-layer chromatography (HPTLC)-based phytochemicals assessment of Drakshavaleha was done quantitatively and qualitatively. This is the very first report on the quantification of biologically active compounds in Drakshavaleha using the HPTLC technique. The study reflects its ease of phytochemicals identification, flexibility, reliability, and simplicity, in the formulation of Drakshavaleha. Four major bioactive compounds—gallic acid, piperine, quercetin, and resveratrol—were identified in the methanolic extract of Drakshavaleha. The quantities of these marker compounds (quercetin, gallic acid, piperine, and resveratrol) were found to be 1.963 µg/mL, 1.432 µg/mL, 1.436 µg/mL, and 1.602 µg/mL, respectively. In nutshell, Drakshavaleha is a satisfactory source of the abovementioned phytochemicals which is responsible for its potent curative application and traditional use. Standardization of Ayurvedic formulation is a crucial and necessary concern to rationalize their employment in public health and to be contemplated for the reliability of safety and therapeutic efficacy.