In addition to their involvement in tissue remodelling, matrix metalloproteinases (MMPs), in the oral environment, are linked to leakage in the resin-dentine interface through their involvement in the proteolytic degradation of the resin-dentine hybrid layer. Numerous studies have evaluated dentine MMP activity and the vast majority of those studies have used an MMP extraction protocol to semi-purify the MMPs from dentine, first described around 20 years ago. This is a protocol that requires 32 days for completion. The technique is based on the three-step sequential use of NaCl solution for pulverised dentine washing, then guanidine HCl and EDTA to demineralise the pulverised dentine to extract the MMPs. In this study, a new one-step dentine MMP extraction protocol was adapted to extract dentine MMPs in only four days. This was achieved by eliminating the NaCl washing step and combining the guanidine HCl and EDTA into one extraction solution that also contained proteinase inhibitors. Fifty-two dentine MMP extracts were obtained utilising the two different extraction methods. The amount of total and endogenously active MMP-2 in the specimens was assayed utilising a human MMP-2 activity enzyme-linked immunosorbent assay (ELISA). The study results showed that the new extraction method is as effective as the traditional three-step extraction method in semi-purifying dentine MMP-2.