Urinary 1,6-hexamethylene diamine (HDA) has been indicated as a potential biomarker for systemic exposure to 1,6-hexamethylene diisocyanate (HDI). However, the quantitative relationships between dermal and inhalation exposure to HDI and urine HDA levels have not been established. The objective of this research was to establish a quantitative and time-dependent relationship between urine HDA levels and inhalation and dermal exposure to HDI. Urine HDA levels, creatinine concentrations, and specific gravity along with dermal and breathing-zone levels of HDI were measured over the course of an entire workday for up to three separate workdays spaced approximately one month apart in 48 automotive spray painters. Dermal exposure and inhalation exposure levels, adjusted for the type of respirator worn, and creatinine concentration were observed to be significant predictors of urine HDA levels. Our results indicate that HDA exhibits a biphasic elimination pattern, with a half-life of 2.9 h for the fast elimination phase. The use of coveralls, the type of respirator worn, smoker status, the type of painting booth, and multiple painters at the shop were all found to significantly affect the creatinine concentration adjusted urine HDA levels. Our results indicate that urine HDA is, in fact, a useful biomarker to investigate systemic HDI exposure through both the skin and the lungs and, further, emphasize the need for proper dermal and inhalation protection for workers in this industry. Further, our results will help industrial hygienists design a program to use urinary HDA to assess occupational HDI-exposure.