Cellular induction of reductase enzymes can alter the susceptibility of cells toward drugs and chemicals. In this study, we compared the capacity of a single dose of sodium selenite and 3H-1,2-dithiole-3-thione (D3T) to influence the drug-relevant reducing capacity of HT29 cells over time, and defined the protein-specific contribution to this activity on the basis of selected reaction monitoring mass spectrometry. Thioredoxin reductase 1 (TrxR1) protein levels and activity were inducible up to 2.2-fold by selenium. In contrast, selenium had only a minor influence on prostaglandin reductase 1 (PTGR1) and quinone oxidoreductase 1 (NQO1) activity and protein levels. D3T, a strong Nrf2 inducer, induced all the reductases and additionally increased the cytotoxicity of hydroxymethylacylfulvene, a bioreductive DNA-alkylating drug. The data and experimental approaches allow one to define induction potency for reductase enzymes PTGR1, TrxR1, and NQO1 in HT29 cells and link these to changes in drug cytotoxicity.