Simultaneous and accurate measurement of vitamin D and 25‐hydroxyvitamin D in biological samples is a barrier limiting our ability to define “optimal” vitamin D status. Thus, our goal was to optimize conditions and evaluate an LC‐MS method for simultaneous detection and quantification of vitamin D2, vitamin D3, 25‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3 in serum. Extraction and separation of vitamin D forms were achieved using acetone liquid–liquid extraction and by a reversed phase C8 column, respectively. Detection was performed on a triple quadrupole tandem mass spectrometer (QQQ‐MS/MS) equipped with atmospheric pressure photo ionization source. The LOQs for all analytes tested were 1 ng/mL for hydroxylated molecules and 2 ng/mL for the parent vitamin Ds. RSD at lower LOQ (2 ng/mL) and in medium (80 ng/mL) and high (200 ng/mL) quality control samples did not exceed 20 and 15% CV, respectively. Accuracy of the method for determination of hydroxylated molecules was also validated using National Institutes of Standards and Technology standard samples and found to be in the range of 90.9–111.2%. In summary, a sensitive and reproducible method is reported for simultaneous quantification of vitamin D2, vitamin D3, 25‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3 molecules in biological samples.