Lipids play diverse roles in cellular function, and protein-lipid interactions often provide the basis for these functions. Photoaffinity labeling is a powerful approach that can capture protein-lipid interactions in their native environments and enable biochemical characterization and imaging studies. In chapter 1 of this thesis, I summarize recent advances in photoaffinity labeling approaches to study lipid functions, and applied this strategy in chapter 2 to understanding the functions of a phospholipase D-generated lipid metabolite of ethanol that is clinically used as an alcoholism marker. I also harnessed the photocrosslinking ability of this strategy to explore in vivo labeling of phospholipase D activity in chapter 3. Finally, chapter 4 of this thesis provides summary and outlook on photocrosslinking chemical probes of phospholipase D signaling. Collectively, I described diverse applications of this powerful strategy towards understanding phospholipase D signaling in cells and in tissues.