The aim of our study was to investigate cerebrospinal fluid (CSF) tryptic peptide profiles as potential diagnostic biomarkers for the discrimination of parkinsonian disorders. CSF samples were collected from individuals with parkinsonism, who had an uncertain diagnosis at the time of inclusion and who were followed for up to 12 years in a longitudinal study. We performed shotgun proteomics to identify tryptic peptides in CSF of Parkinson’s disease (PD, n=10), multiple system atrophy patients (MSA, n=5) and non-neurological controls (n=10). We validated tryptic peptides with differential levels between PD and MSA using a newly developed selected reaction monitoring (SRM) assay in CSF of PD (n=46), atypical parkinsonism patients (AP; MSA, n=17; Progressive supranuclear palsy; n=8) and non-neurological controls (n=39). We identified 191 tryptic peptides that differed significantly between PD and MSA, of which 34 met our criteria for SRM development. For 14/34 peptides we confirmed differences between PD and AP. These tryptic peptides discriminated PD from AP with moderate-to-high accuracy. Random forest modelling including tryptic peptides plus either clinical assessments or other CSF parameters (neurofilament light chain, phosphorylated tau protein) and age improved the discrimination of PD vs. AP. Our results show that discovery of tryptic peptides by untargeted and subsequent validation by targeted proteomics is a suitable strategy to identify novel potential CSF biomarkers for PD versus AP. Furthermore, the tryptic peptides, and corresponding proteins, that we identified as differential biomarkers may increase our current knowledge about the disease-specific pathophysiological mechanisms of parkinsonism.