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.