建立了同时测定清热解毒口服液中的绿原酸、栀子苷、黄芩苷、连翘苷和靛玉红5种有效成分的高效液相色谱-电喷雾电离质谱(HPLC-ESI/MS)分析方法。采用ZorbaxSBC18色谱柱,以含0.2%甲酸的0.4mmol/L醋酸钠(A相)、乙腈(B相)为流动相进行梯度洗脱,在ESI正离子模式下,采用选择离子监测方法进行测定,用峰面积进行定量。结果表明,绿原酸、栀子苷、黄芩苷、连翘苷和靛玉红的线性范围分别为0.050~50mg/L,0.020~20mg/L,0.005~30mg/L,0.010~15mg/L和0.010~10mg/L;检出限分别为0.010,0.005,0.001,0.002和0.003mg/L。5种成分的加样回收率为97.0%~101.7%,相对标准偏差小于2.2%。该法快捷、准确、重复性好,可用于清热解毒口服液中的5种有效成分含量的同时测定。