Biomonitoring methylene dipheny diisocyanate (MDI) in urine may be useful in industrial hygiene and exposure surveillance approaches toward disease (occupational asthma) prevention, and in understanding pathways by which internalized chemical is excreted. We explored possible urine biomarkers of MDI exposure in mice after respiratory tract exposure to MDI, as glutathione (GSH) reaction products (MDI-GSH), and after skin exposure to MDI dissolved in acetone. LC-MS analyses of urine identified a unique 543.29 m/z [M+H]⁺ ion from MDI exposed mice, but not controls. The 543.29 m/z [M+H]⁺ ion was detectable within 24 hours of a single MDI skin exposure and following multiple respiratory tract exposures to MDI-GSH reaction products. The 543.29 m/z [M+H]⁺ ion possessed properties of di-lysine-MDI, including (a) an isotope distribution pattern for a molecule with the chemical formula C27H38N6O6, (b) expected collision induced dissociation (CID) fragmentation pattern upon MS/MS, and (c) a retention time in reverse phase LC-MS identical to synthetic di-lysine-MDI. Further MDI-specific western blot studies suggest albumin (which contains multiple di-lysine sites susceptible to MDI carbamylation) as a possible source for di-lysine-MDI, and the presence of MDI conjugated albumin in urine up to 6 days post respiratory tract exposure. Two additional [M+H]⁺ ions (558.17 and 863.23 m/z) were found exclusively in urine of mice exposed to MDI-GSH via the respiratory tract and possessed characteristics of previously described cyclized MDI-GSH and oxidized glutathione (GSSG)-MDI conjugates respectively. Together the data identify urinary biomarkers of MDI exposure in mice and possible guidance for future translational investigation.