Cobalamin (vitamin B12) deficiency is a common cause of megaloblastic anemia in Western populations. Laboratory evaluation of megaloblastic anemia frequently includes the assessment of patient cobalamin and folate status. Current total serum cobalamin measurements are performed in the clinical laboratory with competitive binding luminescence assays, whose results may not always accurately reflect actual cobalamin stores. Surrogate markers of cobalamin deficiency such as methylmalonic acid and homocysteine have been utilized to improve diagnostic accuracy; however, the specificity of these tests by themselves is rather low. Measurement of the biologically active fraction of cobalamin, holotranscobalamin, has been proposed as a replacement for current total cobalamin assays. Although holotranscobalamin measurements appear to have slighter better sensitivity, the specificity of this assay remains to be determined. The relative merits and demerits of commonly available methods to assess cobalamin deficiency in patients with suspected megaloblastic anemia are discussed. Am. J. Hematol. 88:522–526, 2013. © 2013 Wiley Periodicals, Inc.