The social amoeba Dictyostelium discoideum is already known as a model organism for a variety of cellular and molecular studies. Now that the genome sequencing project has been completed and different tools with which to overexpress or knock out genes are available, this species has also moved into the spotlight of functional genomics studies. Consequently, this genomic sequence information can now be exploited to realize D. discoideum proteomics projects. Here, we present validated protocols adapted for analysis of the D. discoideum proteome. The workflow described in this chapter comprises two-dimensional polyacrylamide gel electrophoresis for protein separation and peptide mass fingerprint (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) for protein identification.