The chemical profile of five main bioactive constituents including rutin, quercetin 3-O-β-D-glucuronopyranoside, quercetin 3-O-β-D-glucopyranosyl(1→2)-O-α-L-rhamnopyranoside, quercitrin and sauchinone in extract of Saururus chinensis was studied using high performance liquid chromatography-diode array detector-electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS). The HPLC-DAD conditions were optimized for the simultaneous analysis of these five compounds. This method was validated in terms of specificity, linearity (R2 > 0.9987), precision (<6.4% RSD), and recoveries (93.9 − 103.0%). The limits of detection of these compounds were ranged from 38.7 to 648.4 ng. In addition, the seasonal variation of the chemical composition of these main compounds in S. chinensis was investigated using this validated method, which resulted in a significant difference in the contents of these compounds according to the harvest time.