Cells respond to external stimuli by transducing signals through a series of intracellular molecules and eliciting an appropriate response. The cascade of events through which the signals are transduced include post-translational modifications such as phosphorylation and ubiquitylation in addition to formation of multi-protein complexes. Improvements in biological mass spectrometry and protein/peptide microarray technology have tremendously improved our ability to probe proteins, protein complexes, and signaling pathways in a high-throughput fashion. Today, a single mass spectrometry-based investigation of a signaling pathway has the potential to uncover the large majority of known signaling intermediates painstakingly characterized over decades in addition to discovering a number of novel ones. Here, we discuss various proteomic strategies to characterize signaling pathways and provide protocols for phosphoproteomic analysis.