Alcohol testing is an expanding area of interest due to the impacts of alcohol abuse that extend well beyond drunk driving. However, existing approaches such as blood and urine assays are hampered in some testing environments by biohazard risks. A noninvasive, in vivo spectroscopic technique offers a promising alternative, as no body fluids are required. The purpose of this work is to report the results of a 36-subject clinical study designed to characterize tissue alcohol measured using near-infrared spectroscopy relative to venous blood, capillary blood, and breath alcohol. Comparison of blood and breath alcohol concentrations demonstrated significant differences in alcohol concentration [root mean square of 9.0 to 13.5 mg/dL] that were attributable to both assay accuracy and precision as well as alcohol pharmacokinetics. A first-order kinetic model was used to estimate the contribution of alcohol pharmacokinetics to the differences in concentration observed between the blood, breath, and tissue assays. All pair-wise combinations of alcohol assays were investigated, and the fraction of the alcohol concentration variance explained by pharmacokinetics ranged from 41.0% to 83.5%. Accounting for pharmacokinetic concentration differences, the accuracy and precision of the spectroscopic tissue assay were found to be comparable to those of the blood and breath assays.