|Recently, low serum levels of 25-hydroxy vitamin D3 and D2 along with metabolites of vitamin D, have been linked to a number of conditions including; hypertension, autoimmune diseases, and cancer. This has driven significant increases in testing volume for these analytes at clinical laboratories. Accurate and rapid analysis of 25-hydroxyvitamin D is essential for assessing vitamin D status and determining its role in various diseases. Immunoassays have traditionally been used in clinical settings, but suffer from inherent methodological problems including decreased dynamic range and specificity. We present a sensitive, accurate, and robust high-throughput multiplexed assay for 25-hydroxyvitamin D using LC-MS/MS.