Deficiencies of folate or of vitamin B-12 are widespread and constitute a major global burden of morbidity that affect all age groups. Detecting or confirming the presence of folate or vitamin B-12 deficiency and distinguishing one from the other depends, ultimately, on laboratory testing. Tests to determine the presence of folate or vitamin B-12 deficiency are used singly or in combination to establish the nutritional status and prevalence of deficiencies of the vitamins in various populations. The efficacy of interventions through the use of fortification or supplements is monitored by using the same laboratory tests. Tests currently in use have limitations that can be either technical or have a biological basis. Consequently, each single test cannot attain perfect sensitivity, specificity, or predictive value. Laboratory indicators of vitamin B-12 or folate status involve the measurement of either the total or a physiologically relevant fraction of the vitamin in a compartment such as blood. Thus, assays to measure vitamin B-12 or folate in plasma or serum as well as folate in red blood cells are in widespread use, and more recently, methods to measure vitamin B-12 associated with the plasma binding protein transcobalamin (holotranscobalamin) have been developed. Alternatively, concentrations of surrogate biochemical markers that reflect the metabolic function of the vitamin can be used. Surrogates most commonly used are plasma homocysteine, for detection of either vitamin B-12 or folate deficiency, and methylmalonic acid for detection of vitamin B-12 deficiency. The general methods as well as their uses, indications, and limitations are presented.