Cheilanthes albomarginata Clarke, a fern from the Pteridaceae family, is traditionally used to treat peptic ulcer and stomach disorder. Six flavonoid compounds were isolated and identified from the ethyl acetate soluble fraction of this plant. Kumatakenin 5-O-β-glucoside,kaempferol-7-methyl ether, rhamnocitrin 3-O-β-glucoside, and kaempferol 3-O-β-glucoside were isolated for the first time from this plant. UPLC method was developed and used for the simultaneous determination and quantification of the isolated compounds in the methanol extract of the plant. The method was also validated using the parameters: linearity, precision, and accuracy.