Updated project metadata. The fatal neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are the most common motoneuron disease and genetic cause of infant death, respectively. Various in vitro model systems have been established to investigate motoneuron disease mechanisms - in particular immortalized cell lines and primary neurons. By quantitative mass spectrometry (MS)-based proteomics we here compare the proteomes of primary motoneurons to motoneuron-like cell lines NSC-34 and N2a as well as to non-neuronal control cells at a depth of 10,000 proteins. We use this resource to evaluate the suitability of murine in vitro model systems for cell biological and biochemical analysis of motoneuron disease mechanisms. Individual protein and pathway analysis indicate substantial differences between motoneuron-like cell lines and primary motoneurons, especially for proteins involved in differentiation, cytoskeleton and receptor signaling, whereas common metabolic pathways were more similar. The ALS-associated proteins themselves also showed distinct differences between cell lines and primary motoneurons, providing a molecular basis for understanding fundamental alterations between cell lines and neurons with respect to neuronal pathways with relevance for disease mechanisms. Our study provides a proteomics resource for motoneuron research and presents a paradigm of how MS-based proteomics can be used to evaluate disease model systems