Vitamin B 12 deficiency is widespread. Among the population groups at risk are older people, vegetarians, pregnant women and patients with renal or intestinal diseases. The neurological symptoms of vitamin B 12 deficiency are unspecific and can be irreversible. Early detection is therefore important. This article reviews the diagnostic performance of the different laboratory markers for vitamin B 12 status. Total serum vitamin B 12 is a relatively insensitive and unspecific biomarker of deficiency that does not reflect recent variations in cobalamin status. Holotranscobalamin (holoTC), the metabolically active portion of vitamin B 12 , is the earliest laboratory parameter that becomes decreased in case of a vitamin B 12 negative balance. Concentration of methylmalonic acid (MMA) is a functional vitamin B 12 marker that will increase when the vitamin B 12 stores are depleted. Isolated lowering of holoTC shows vitamin B 12 depletion (negative balance), while lowered holoTC plus elevated MMA (and homocysteine) indicates a metabolically manifested vitamin B 12 deficiency, although there still may be no clinical symptoms. The diagnostic use of holoTC allows the initiation of therapeutic measures before irreversible neurological damage develops. Because the clinical manifestations of vitamin B 12 deficiency are unspecific, people at risk should be identified and should regularly test their holoTC with or without MMA.