Gingipains (RgpA, RgpB, and Kgp) are major virulence factors of the periodontitis-causing bacterium Porphyromonas gingivalis. Isolation of gingipains from the crude protein sample of P. gingivalis is critical for studying the underlying invasion mechanism that contributes to periodontitis, Alzheimer’s disease, and cardiovascular disease (CVD). Chromatographic processes and molecular cloning are two standard techniques often used for gingipains isolation, which are time-consuming and costly. In this study, considerably easier methods based on passive-mediated diffusion gel elution and gelatin zymogram were used to isolate and characterize gingipains. Importantly, proteins eluted from Native-PAGE showed enzymatic activity for both Rgp and Kgp. In gelatin zymography, the proteins with a molecular size of ~50 kDa and above 245 kDa were suggested as arginine-specific gingipains. The passive diffusion-mediated gel elution method is a simpler technique to isolate gingipains from crude protein samples of P. gingivalis. By using covalent and highly specific gingipain inhibitors, gelatin zymography enabled an individual characterization of gingipain activity and inhibition. Finally, this protocol can be easily extended by adding the isoelectric focusing to further improve the protein separation and characterization.