Sphingosine 1-phosphate (S1P), a bioactive lipid involved in various physiological processes such as cell proliferation and apoptosis, can be irreversibly cleaved by the S1P lyase, yielding phosphoethanolamine and (2E)-hexadecenal (2EHD). The latter metabolite, an α,β-unsaturated fatty aldehyde, may be susceptible to nucleophilic attack by cellular biomolecules. Hence, we studied whether 2EHD forms reaction products with glutathione (GSH) and proteins in vitro. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and stable isotopically labeled reference material, we identified a total of nine novel reaction products of 2EHD in a cell-free approach: two GSH conjugates and seven L-amino acid adducts. Both GSH conjugates were also found in HepG2 cell lysates incubated with 2EHD. Likewise, we detected 4 out of 7 amino acid adducts released from the model protein BSA and proteins extracted from HepG2 cells. Hereby, the 2EHD Michael adduct with L-histidine proved to be the most prominent adduct. Most interestingly, inhibition of the enzymatically driven oxidative degradation of 2EHD resulted in increased levels of both GSH conjugates and protein adducts in HepG2 cell lysates. Hence, our data provide new insights into sphingolipid metabolism and will be useful to investigate certain disorders linked to an impaired fatty aldehyde metabolism in more detail.