Background: National survey data of erythrocyte glutathione reductase activation coefficient (EGRac) indicate that suboptimal riboflavin status may be a problem in all population age groups, but the cutoff for deficiency is controversial. In addition, the effectiveness of different biomarkers of riboflavin status has not been critically evaluated. Objective: We aimed to assess the effectiveness of different biomarkers of riboflavin status through a systematic review of published riboflavin supplementation trials. Design: We structured our search strategy on Ovid MEDLINE, EMBASE (Ovid), and Cochrane databases; formal inclusion and exclusion criteria; data extraction; validity assessment; and meta-analysis. Results: Eighteen supplementation studies reporting up to 14 biomarkers were included. Sufficient data were available to show that EGRac (14 studies) and basal glutathione reductase activity (5 studies) were effective biomarkers of altered riboflavin intake (P < 0.00001), although substantial heterogeneity (I-2 > 66%) that could not be explained by the subgroup analysis was observed. Plasma total homocysteine was not an effective biomarker of riboflavin status in the general population, but some evidence identified its potential usefulness specifically in those homozygous for a common polymorphism in the MTHFR gene. Conclusions: The evidence suggests that EGRac is an effective biomarker of a change in riboflavin intake in populations with severe-to-normal baseline status. Studies of healthy populations that compare the response to low-dose supplementation among different age, sex, and MTHFR genotype groups are required to provide evidence for generating dietary riboflavin recommendations specific to different population subgroups. Further research into alternative biomarkers to EGRac is also required. Am J Clin Nutr 2009; 89 (suppl): 1960S-80S.