Abstract   Tripeptidyl-peptidase II (TPPII), is present in most eukaryotic cells. It cuts tripeptides from the N-terminus of peptides and is especially important for degrading peptides longer than 15 amino acids. TPPII also tailors long peptides into suitable substrates for the enzymes which transport and produce the peptides that MHC I present. Increased levels of TPPII have also been found in certain cancer cells, thus it is of interest to determine if TPPII could be used as a tumour marker. The aim of this study was to optimise and evaluate 3 different methods for quantifying TPPII. Western blot, enzyme-linked immunosorbent assay (ELISA) and fluorophore-linked immunosorbent assay (FLISA) protocols were optimised regarding incubation times and antibody dilutions. Sensitivity and linearity were the most important parameters when evaluating the results. The coefficient of determination of western blot was R2=0.98-1 within the range of 1.29-250ng TPPII/well and ELISA had a coefficient of determination of R2=0.96 within the range of 0.03-250ng TPPII/well. Presently western blot is the only one of these methods to yield reliable results with impure samples, but ELISA is superior regarding sensitivity and throughput. Thus further optimisation of ELISA is interesting to pursue.