Objective A method using high performance liquid chromatography tandem mass spectrometry for simultaneous determination of folic acid and biotin in compound vitamin pills was presented. Methods Samples were extracted by water. The analysis was carried out on an Agilent Eclipse C₁₈(2.1 mm×100 mm,1.8 μm)column with isocratic elution using a mobile phase of 0.1% aqueous formic acid and acetonitrile at 0.2 ml/min. Mass spectrometry acquisition was done in the positive ion mode and the analytes were detected in the multiple reaction monitoring(MRM)mode. Results Folic acid and biotin in samples could be well separated within 5 min. The calibration cures were linear over the concentration range of 42-523 ng/ml and 47-583 ng/ml for folic acid and biotin, respectively. The linear regression equations were y=1157.63x-6698.38(r²=0.999 9) and y=207.93x-3052.26(r²=0.998 2), respectively. The average recoveries were 99.9%-108.7% and 90.1%-94.5% for folic acid and biotin at low, medium and high adding levels, respectively. The relative standard deviation(RSD)were both 1.0% (n=6). The detection limit was 0.33 and 0.08 μg/g for folic acid and biotin, respectively. Conclusion The developed method was precise, accurate and sensitive, which therefore could be applied for the analysis of folic acid and biotin in compound vitamin pills.