An important component of nephrology research is the discovery of novel proteins that control cellular and molecular events that contribute to normal kidney cell biology and disease. Identifying perturbation of normal cellular protein expression and interactions within signaling networks is critical for understanding these regulatory events. Methods that couple 2-dimensional capillary liquid chromatography and tandem mass spectrometry (2D-LC-MS/MS) analysis have greatly facilitated this discovery science. Coupling 2D-LC-MS/MS analysis with automated genome-assisted spectra interpretation allows a direct, high-throughput, and high-sensitivity identification of hundreds to thousands of individual proteins from targeted complex biological samples. The systematic qualitative and quantitative comparison of experimental/disease conditions and appropriate controls allow protein function or disease states to be modeled. This review discusses the different purification and quantitative strategies that have been developed and used in combination with 2D-LC-MS/MS and computational analysis to define regulatory events in kidney biology and disease.