High-throughput separations are intrinsic to the detection and analysis of peptides and proteins by mass spectrometry (MS). Together, efficient separation and MS can lead to the identification of thousands of proteins in a sample, cell or tissue and help build proteome maps that can be used to define a cell type or cellular state. Although 2D gels have been successfully used to separate proteins for subsequent MS analysis, alternative separation efficiencies and, consequently deeper results could be obtained with HPLC or other separation techniques that improve throughput. This highlight is aimed toward plant scientists who have special separation needs due to the nature of plant cells and who could benefit from knowing options and requirements for adopting alternative separation protocols. Through the various sample processing and protein separation strategies, plant biologists should be able to improve the quality of their proteomic reference maps and gain new information about the proteins that define plant cells.