A laboratory experiment for teaching protein separation and detection in an undergraduate biochemistry laboratory course is described. This experiment, performed in two, 4 h laboratory periods, incorporates guided inquiry principles to introduce students to the concepts behind and difficulties of protein purification. After using size-exclusion chromatography to separate a mixture of proteins, students utilize a colorimetric enzymatic assay and an immunoassay to determine the location of individual mixture components. Students proceed to determine the molecular weight of each protein using gel electrophoresis and generated mass spectrometric data. Completing this experiment provides students with an opportunity to expediently separate proteins while learning about protein characterization.