Abstract Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, and novel biomarkers are needed. We applied mass-spectrometry-based peptidomic analysis in cerebrospinal fluid (CSF) samples of ALS and non-neurodegenerative control patients (Con) from a discovery (n = 48) and validation (n = 109) cohort for biomarker discovery. Systematic selection revealed a panel of eight novel peptide biomarker candidates for ALS (out of 33,605) derived from seven proteins. In the validation cohort, NFL, MAP1B, MYL1, and APOC1 peptides were upregulated, and peptides from CADM3, SCG1, and PENK were downregulated in ALS compared to Con. The peptides (except NFL) were not changed in other neurodegenerative diseases, including Alzheimer´s disease, frontotemporal dementia and Parkinson´s disease. Combination of all peptides in a logistic regression model led to an area under the curve value of 98% for the discrimination of ALS from controls. Data of the NFL peptide strongly correlated with an established NFL immunoassay (Ella, r = 0.97). The peptide biomarker candidates are derived from proteins with different function, and their determination with our method provides the opportunity for simultaneous investigation of key processes in ALS.