Vitamin B12 levels are tested in different ways with different accuracies. Serum B12 by radioimmune assay (RIA) is less accurate than microbial assay since it picks up all forms of cobalamin including those that are inactive. Serum B12 by microbial assay appears to be the most widely used and is considered accurate. However, pregnancy, large doses of vitamin C, and folate deficiency may result in a falsely reduced B12 microbial assay. A 24 hour urine MMA (Methylmalonic acid) test is claimed to be very sensitive but is also more expensive. Without B12, MMA increases in the urine.

Another testing pattern used by doctors to determine vitamin B12 status is to test for intrinsic factor (IF) antibodies. Their presence prevents the normal binding of B12 to IF and thus prevents B12 absorbtion. Of pernicious anemia sufferers, 70% have these antibodies. If the test for IF antibodies is negative, a Schilling test is usually performed to help distinguish the nature of the problem. This test involves the use of radioactively labelled B12 and may be objectionable to some people.

A lack of gastric hydrochloric acid tends to confirm a suspected lack of intrinsic factor (IF), as both can be due to a shrinking of gastric cells.