Human noroviruses (HuNV) are members of the Caliciviridae family and are a significant cause of gastroenteritis worldwide. Cell culture models are challenging for studying the intracellular replication of HuNV, with murine norovirus (MNV) routinely used to study norovirus replication. Norovirus genomes contain a non-structural (NS) polyprotein that is processed by the viral protease generating precursor and mature proteins during replication. We identified a putative viral protease cleavage site at E66/G67 in the MNV-1 NS1-2 protein by sequence analysis that was cleaved by the precursor ProPol but not Pro in vitro and during MNV-1 replication. We used mass spectrometry to confirm the cleavage site and the protease activity being specific to ProPol but not Pro. We used the TAILS method (Kleifeld et al. Nat Protoc. 2011;6(10):1578-611) with iTRAQ labelling to identify new N-termini after protease cleavage and data-dependent acquisition mass spectrometry as well as parallel reaction monitoring of tryptically digested gel bands of cleavage products to confirm the new N-terminus at E66/G67 in the MNV-1 NS1-2 protein.