Complex phytochemical composition of polyherbal formulations which are dominant in traditional medicine systems makes it hard to standardize them. The research discusses a confirmed analytical method of High-Performance Thin-Layer Chromatography (HPTLC) and Ultraviolet-Visible (UV-Vis) Spectroscopy to standardize and guarantee the quality control of polyherbal formulation. Validated HPTLC methods based on ICH guidelines were used to quantify selected marker compounds-gallic acid, quercetin, mangiferin and curcumin- in terms of linearity, accuracy, precision, specificity, limits of detection (LOD) and limits of quantification (LOQ). In tandem with this, UV-Vis absorption spectra and calibration curves were developed to quantitatively analyse substances using Beer Lambert law with the instrumental limitations in mind (e.g. stray light and bandwidth of the dual wavelength approach). These results obtained high correlation coefficients (>0.999), detection limit in nanogram range and accurate quantification of significant bioactive markers. The combined workflow provides a sound, reproduceable and cost-efficient platform of quality control of complex herbal blends. The study helps to advance the use of polyherbal preparations because it helps to mediate between traditional medicine approaches and contemporary analytical standards.