To comprehend applied termite research, advances in our knowledge of fundamental termite biology are required. This study was conducted to establish methods for characterizing the Reticulitermes flavipes proteome and provide a basis for future R. flavipes protein research. Termites from different colonies of R. flavipes were collected from Stillwater, OK. and maintained in the laboratory for a minimum of 30 days. Termites were harvested and converted to a whole-body termite protein extract. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to separate the proteins. Visualization of the gels was completed using Coomassie brilliant blue staining and silver staining techniques. A protein map of the resulting protein spot pattern was generated by numbering each spot and assigning Cartesian coordinate measurements correlating to isoelectric point (pI) and molecular weight (MW). After mapping, R. flavipes protein characterization was initiated using matrix assisted laser desorption/ionization – time of flight (MALDI-TOF) mass spectrometry to generate peptide mass fingerprints (PMFs). Protein identification was initiated by comparing the PMF against various databases for a putative identification. Next, n-terminal sulfonation of the peptides was used to provide chemically assisted fragmentation. Sulfonated fragments were analyzed using MALDI-TOF to elucidate amino acid sequence data to verify the accuracy of the R. flavipes protein identification. This initial study of the proteome will facilitate future research among R. flavipes as well as with other termite species.