Assessment of the different conformational states of the abnormal prion protein (PrPSc) in the CNS provides an established basis for distinguishing transmissible spongiform encephalopathy (TSE) strains. PrPSc conformers are variably resistant to N-terminal proteinase K (PK) digestion, and analysis of the consensus products (PrPres) by immunoassay enables effective, but relatively low-resolution differentiation. Determination of the precise N-terminal amino acid profile (N-TAAP) of PrPres presents a potential high-resolution means of TSE-strain typing, and thus of differential disease diagnosis. This approach was evaluated using individual mice affected by model scrapie (22A, ME7, 87V and 79A) and bovine spongiform encephalopathy (BSE) (301V) strains. Nano liquid chromatography–mass spectrometry (LC-MS) was used to determine PrPres N-terminal tryptic digestion products. Four major N-terminal tryptic peptides were generated from all mouse TSE strains investigated, corresponding with predominant N-termination of PrPres at G81, G85, G89 and G91. Both the mass spectrometric abundance of the individual peptides and the ratios of pairs of these peptides were evaluated as markers of conformation in relation to their potential for strain discrimination. The yield of peptides was significantly greater for BSE than scrapie strains and the relative quantities of particular peptide pairs differed between strains. Thus, whereas peptide G91–K105 was a dominant peptide from 301V, this was not the case for other strains and, significantly, the ratio of peptides G91–K105:G89–K105 was substantially higher for BSE-infected compared with scrapie-infected mice. These data support the potential of the N-TAAP approach for high-resolution TSE strain typing and differential diagnosis.