Biomonitoring of methylene diphenyl 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 the 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 m/ z 543.29 [M + H]+ ion from MDI-exposed mice but not from controls. The m/ z 543.29 [M + H]+ ion was detectable within 24 h of a single MDI skin exposure and following multiple respiratory tract exposures to MDI-GSH reaction products. The m/ z 543.29 [M + H]+ ion possessed properties of dilysine-MDI, including (a) an isotope distribution pattern for a molecule with the chemical formula C27H38N6O6, (b) the expected collision-induced dissociation (CID) fragmentation pattern upon MS/MS, and (c) a retention time in reversed-phase LC-MS identical to that of synthetic dilysine-MDI. Further MDI-specific Western blot studies suggested albumin (which contains multiple dilysine sites susceptible to MDI carbamylation) as a possible source for dilysine-MDI and the presence of MDI-conjugated albumin in urine up to 6 days after respiratory tract exposure. Two additional [M + H]+ ions ( m/ z 558.17 and 863.23) 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.