Background: Riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are cofactors in oxidation-reduction reactions. The aim of this study was to compare a direct high performance liquid chromatography (HPLC) method with an indirect enzymatic method to assess flavin status and establish reference intervals. The HPLC method was used to assess prevalence of flavin deficiency in total parenteral nutrition (TPN) patients. Method: Blood was obtained from 90 healthy volunteers and 51 TPN patients. Results for each method were compared using Spearmans Rank correlation. Results from reference groups and TPN patients were compared using Mann-Whitney U tests. Results: Erythrocyte glutathione reductase activity coefficients (EGRAC) correlated with flavin levels measured by HPLC (p<0.001). HPLC was chosen for TPN samples analyses due to superior sensitivity. There was no significant difference between FAD and FMN levels in controls and TPN patients, although TPN patients had significantly higher levels of riboflavin than controls (p<0.001). Conclusion: HPLC is superior to the EGRAC assay for assessing flavin status. Hyporiboflavinosis is not prevalent in TPN patients.