•Glyoxylic acid preferentially modifies lysine over arginine residues in BSA.•Glyoxal alone (300 mM) can convert up to 24% of BSA lysine into CML.•Glyoxal forms more fluorescent products in BSA compared with glyoxylic acid.•In rodent food fortified with glycated-BSA, CML declined 30% over 80 days storage.•33% of dCML from glycated-BSA fortified food was excreted in mouse feces. This study investigated different methods to produce Nε-carboxymethyl-lysine (CML)-enriched bovine serum albumin (BSA) as alternatives to the classical approach using glyoxylic acid (GA) and sodium cyanoborohydride (NaBH3CN) which results in toxic hydrogen cyanide (HCN). The reaction of GA (6 mmol/L) and NaBH3CN (21 mmol/L) to produce CML remained the most effective with CML yields of 24–35%, followed by 13–24% using 300 mmol/L glyoxal (GO). GA promoted specific modification of lysine to CML, and fewer structural modifications of the BSA molecule compared with GO, as evidenced by fluorescence and proteomic analyses. GO promoted greater arginine modification compared with GA (76 vs 23%). Despite structural changes to BSA with GO, murine fecal clearance of CML was similar to literature values. Hence, BSA glycation with 300 mmol/L glyoxal is a suitable alternative to GA and NaBH3CN for generating CML-enriched protein free of HCN, but a CML-only fortification model remains to be described.