Biological monitoring of 1,6-hexamethylene diisocyanate (HDI) exposure has been limited to the hydrolysis product of HDI monomer. The determination of acetylated 1,6- diaminohexane (HDA) and isotriamine as biomarkers of HDI and isocyanurate exposure, respectively, can provide important insight into the metabolism and dose-response relationships of these compounds. The objective of this study was to develop a liquid chromatography-mass spectrometry (LC-MS) method to detect HDA, monoacetyl-HDA, diacetyl-HDA, and isotriamine in urine. The LC-MS method was developed using the four standards spiked into urine at concentrations between 0.019-10.144 μg/L. Quantification of serial dilutions of the standards in urine was performed using selected reaction monitoring on a triple quadrupole mass spectrometer. The limit of detection and the limit of quantification in spiked urine were 0.01 and 0.02 μg/L for monoacetyl-HDA, respectively, and 0.16 and 0.31 μg/L for diacetyl-HDA, respectively. HDA and isotriamine were not detectable in urine at concentrations below 10 μg/L because of high background level of other unspecified compounds. The developed LC-MS method is sensitive and specific for the quantification of acetylated metabolites of HDI. However, modifications to the method are necessary to improve the specificity and sensitivity of HDA and isotriamine analyses.