The inflammatory process underlying chronic obstructive pulmonary disease (COPD) may be caused by tobacco smoke (TS) exposure. Previous studies show that epoxyeicosatrienoic acids (EETs) possess promising anti-inflammatory properties, therefore stabilization of EETs and other fatty acid epoxides through inhibition of soluble epoxide hydrolase (sEH) was investigated in mouse models of acute and sub-chronic inflammation caused by TS exposure. During the entire TS exposure, the potent sEH inhibitor 1-(1-methylsulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea (TUPS) was given via drinking water. To assess drug target engagement of TUPS, a tandem mass spectrometry method was used for bioactive lipid profiling of a broad range of fatty acid metabolites, including EETs, and their corresponding diols (DHETs) derived from arachidonic acid, as well as epoxides and diols derived from other fatty acids. Several, but not all, plasma epoxide/diol ratios increased in mice treated with sEH inhibitor, compared to non-treated mice suggesting a wider role for sEH involving more fatty acid precursors besides arachidonic acid. This study supports qualitative use of epoxide/diol ratios explored by bioactive lipid profiling to indicate drug target engagement in mouse models of TS exposure relevant to COPD, which may have ramifications for future therapeutic interventions of sEH.