OBJECTIVE： To establish a method for simultaneous determination of 6 antitumor active constituents in Folium isatidis. METHODS： HPLC method was adopted. The determination was performed on Waters XTERRA® MS C18 column with mobile phase consisted of methanol-0.1％ formic acid （gradient elution） at the flow rate of 1.0 mL/min. The column temperature was 35 ℃， and detection wavelength was 254 nm. The injection volume was 10 μL. Using luteolin as internal standard， relative correction factors （RCF） of uridine， adenosine， hyperin， 7-methoxycoumarin and apigenin were calculated respectively. The contents of above 5 antitumor active components in F. isatidis were calculated by RCF. The contents of 6 components were determined by external standard method （ESM）. The results of content determination by QAMS were compared with ESM. RESULTS： The linear range was 0.122 6-4.90 2 μg for uridine， 0.063 4-2.534 μg for adenosine， 0.032 7-1.309 μg for luteolin， 0.038 2-1.527 μg for hyperin， 0.032 3-1.291 μg for 7-methoxycoumarin， 0.052 5-2.098 μg for apigenin（r≥0.999 1）. The limits of detection were 3.06， 1.58， 0.82， 0.96， 0.81， 1.31 μg/mL， respectively. The limits of quantification were 10.21， 5.28， 2.73， 3.18， 2.69， 4.37 μg/mL. RSDs of precision， stability （24 h） and reproducibility tests were less than 2％ （n＝6）. The average recoveries were 96.45％-99.14％ （RSDs were 0.8％-1.6％， n＝6）. RCFs of uridine， adenosine， hyperin， 7-methoxycoumarin and apigenin were 0.197， 0.413， 0.732， 0.825 and 0.587， respectively. RCFs repeatability was perfect under different experiment conditions. There was no significant difference between the quantitative results of the QAMS and ESM （P＞0.05）. CONCLUSIONS： QAMS method is simple， fast， accurate and feasible， and can be used for simultaneous determination of uridine， adenosine， luteolin， hyperin， 7-methoxycoumarin and apigenin in F. isatidis.