Cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2) were determined simultaneously by selected reaction monitoring (SRM) mass spectrometry for different food matrixes. A small amount of starting sample was saponified and extracted before injection into a linear ion trap mass spectrometer equipped with an atmospheric pressure chemical ionization source. Dihydrotachysterol, which is absent from food and has a structure similar to that of vitamins D3 and D2, was used as an internal standard. Calibration curves for the 2 vitamins showed linearity with R2 values of 0.9999 and 0.9989 for vitamins D3 and D2, respectively. Limits of detection for vitamins D3 and D2 were 0.5 ng/g (1.3 pmol/g) and 1.75 ng/g (4.4 pmol/g) and limits of quantitation were 1.25 ng/g (3.24 pmol/g), and 3.75 ng/g (9.45 pmol/g), respectively. Accuracy and precision of the method were tested with the infant formula reference standard of the National Institute of Standards and Technology, which showed a relative standard deviation of 6%. Recoveries ranged from 95 to 105%. Several food products were tested with AOAC Method 982.29, which is currently in use for vitamins D3 and D2, and results were comparable within 6%.