A high-performance liquid chromatography method for the quantification of flavonoids from Macrothelypteris viridifrons (Tagawa) Ching, Thelypteridaceae, was developed. The analysis was performed on a C-18 column with a gradient elution of methanol and water at a flow rate of 1 ml/min and a wavelength of 262 nm. All standard calibration curves exhibited good linearity (r(2) > 0.9955) within the test ranges. The precision (intra-day and inter-day tests) revealed relative standard deviation values within the ranges of 0.54-2.03% and 0.84-2.38%, respectively. The recoveries were in the range of 97.51-100.19%. The relative standard deviation values for stability and repeatability were < 5%. The contents of protoapigenin-4 '-O-glucoside and protoapigenone, as a class of unusual flavonoids, were high and varied in the range of 206.5-230.5 mg/g and 255.7-272.3 mg/g, respectively. Graphical Abstract