OBJECTIVE Nutritional status assessment is a critical tool for the identification of nutrient deficiencies or excesses in individual healthcare and epidemiologic screening. Because low but 'normal' status of folate, vitamin B-12, and vitamin B-6 have been associated with an increased risk of chronic diseases, research has focused on defining sensitive indicators of B-vitamin status and on the development and validation of analytical methods for their quantification. RESULTS With the increasing availability and more user-friendly configuration of liquid chromatograph-tandem mass spectrometers (LC-MS/MS), numerous analytical methods for determination of B-vitamin indicators by LC-MS/MS have been developed over the last years. These methods include folate assays for simultaneous determination of numerous folate forms at their specific reduction level. The functional indicators for vitamin B-12 status are plasma methylmalonic acid and total homocysteine and can be measured, either individually or in combination, by high-throughput analysis using LC-MS/MS. Methods for vitamin B-6 status assessment are multianalyte platforms that determine vitamin B-6 forms and functional indicators by the same assay. CONCLUSIONS The high sensitivity, selectivity, and specificity of isotope-dilution LC-MS/MS [and gas chromatography-mass spectrometry (GC-MS)] techniques have allowed the development of reference methods and the creation of multianalyte platforms. The additional convenience of automated sample preparation enables high sample throughput and makes those sensitive methods prospective analytical candidates for larger settings including clinical laboratories.