Streptococcus pyogenes secretes various proteins to the extracellular environment. During infection, these proteins interact with human macromolecules and contribute to pathogenesis. We describe a proteomic approach routinely used in our laboratory to characterize culture supernatant proteins using small-format two-dimensional gel electrophoresis. Proteins are collected after overnight growth of the bacteria in broth media. Compounds that inhibit isoelectric focusing, such as salts, are removed by enzymatic treatment and precipitation with trichloroacetic acid and acetone. Following resuspension in denaturing solution, the proteins are separated by isoelectric focusing using a 7-cm immobilized strip with a pH gradient of 4–7. Subsequently, proteins are further separated with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained with SYPRO Ruby. The small-gel format requires less time, reagents, and smaller culture volumes compared with large-format approaches, while still resolving and detecting a large proportion of the exoprotein fraction.