One of the main goals of comparative cell signaling analyses is the characterization of protein changes between different biological samples, either globally or by targeting specific proteins of interest. Highly accurate and precise strategies are thus required for the relative quantification of proteins extracted from two or more different cell populations. Stable isotope labeling with amino acids in cell culture (SILAC) is a general method for mass spectrometric quantitative proteomics based on metabolic incorporation of stable isotope-labeled amino acids into the cellular protein pool. This method has been applied with great success to a variety of quantitative proteomics problems aimed at gaining further insight into cell signaling pathways. In this chapter, we describe how SILAC can be used for the elucidation of cellular mechanisms, including temporal proteome profiling and the quantitative analysis of the extent of specific posttranslational modifications.