PURPOSE: To describe a method to characterize the gelatinase activity of cultured human periodontal fibroblasts stimulated with Pam3Cys and E. coli LPS, ligands of TLR2 and TLR4 respectively, and by centrifugation of the cultures, simulating an orthodontic force. METHODS: To study MMP-2 activity, primary cultures of human periodontal fibroblasts were stimulated with the addition of TLRs 2 and 4 ligands and the application of mechanical force by centrifugation at 141 x g for 30 min. Supernatant media was collected 24 hours later to perform protein quantification and zymography. RESULTS: MMP-2 activity suffered an increase in cultures co-stimulated with TLRs 2 and 4 ligands alone or with the presence of mechanical force application compared to basal levels. CONCLUSION: Zymography, one of the several methods to study MMPs activities, is a simple, qualitative and efficient method based on electrophoresis of bis-acrylamide gels copolymerized with a protein substrate.