Bovine trypsin is a well-characterized serine protease used as a vital tool in biochemistry, particularly proteomics, as it cleaves peptides or proteins, with high specificity, at the carboxy-terminus of lysine (Lys) and arginine (Arg) amino acid residues. Moreover, the fragment peptides are typically in mass ranges that are amenable to detection by mass spectrometry techniques other than Matrix Assisted Laser Desorption Ionization (MALDI). In addition, Trypsin is found in the digestive system of many vertebrates, and is stable under diverse chemical conditions. The RAS (Rat Sarcoma) families of proteins are involved in several cell-signaling activities that are vital to processes such as cell proliferation and differentiation. If not properly regulated, these signaling processes can result in diseases such as cancer, underlying their importance as targets for drug discovery efforts to control Ras-stimulated abnormal cell signaling. The activity of Ras-related proteins is dictated by their nucleotide-bound state where the GTP-bound is active, and the GDP-bound state inactive. The regulation of this nucleotide-cycling activity is dictated by protein-protein interactions with effectors that may induce conformational changes. Here, we highlight recent advancements showing the use of trypsin in the characterization of Ras-related protein structure and function in cell signaling and regulation.