Polygonatum is widely used as a part of food in different regions of the world which covers five main categories such as drinks, vegetables, snacks, staple and seasoning foods. Presently, no analytical method is available for the quality control of Polygonatum.Development and validation of a method using ultrahigh-performance liquid chromatography diode array detector quadrupole time-of-flight (UHPLC-DAD/QTOF) technique for the estimation of six compounds including a flavonol glycoside [rutin (1)], two flavonols [quercetin (2) and kaempherol (3)] and three homoisoflavonoids [5,7-dihydroxy-3-(2-hydroxy-4-methoxybenzyl)-chroman-4-one (4), 5,7-dihydroxy-3-(2-hydroxy-4-methoxybenzyl)-8-methylchroman-4-one (5) and 5,7-dihydroxy-3-(4-methoxybenzyl)-8-methylchroman-4-one (6)]. In addition, screening of extract, fractions and compounds of P. verticillatum for antioxidant activity was also determined.The separation was achieved on C-18 column using acetonitrile and water containing 0.1% formic acid. The method was validated as per ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) guidelines. The validated method was applied for the simultaneous identification and quantification of compounds 1-6 in extract (E) and fractions (F1-F4) of P. verticillatum. Furthermore, antioxidant potential of E, F1 and F2 and compounds was evaluated using DPPH (2,2-diphenyl-1-picrylhydrazyl) assay.The method was within the linear range (r ) of 0.982 to 0.999, precise (intra- and inter-day percentage relative standard deviations < 2.72 and 2.26) and accurate with recoveries (89.1-98.3%). The limit of detection and limit of quantification were in the ranges 0.02-0.16 and 0.06-0.48 ng/mL, respectively. Compounds 1-6 were quantified in all the samples. Compounds 1, 2 and 5 showed higher activity with half maximal inhibitory concentration (IC ) values 0.41, 0.39, 0.72 at 10, 20 and 30 μg/mL, respectively.Developed method will be helpful to assess the quality of P. verticillatum raw material and their derived products.