Phenotypic and compositional changes of immune cells in cerebrospinal fluid (CSF) can be used as biomarkers to help diagnose and track disease activity for neuroinflammatory and neurodegenerative diseases, such as multiple sclerosis (MS) and Alzheimer´s disease (AD). The CSF is the compartment in closest proximity to the central nervous system (CNS) with unique immune cell composition and routine CSF collection is integral for diagnosing various CNS disorders, offering a more practical alternative to assess the CNS. In this study, we described an end-to-end workflow to perform in-depth single-cell immune profiling using Cytometry by Time-of-Flight (CyTOF) on cells isolated from the CSF of 83 patients with non-neuroinflammatory diseases (n=22), MS (n= 32) and AD (n= 29). We established a streamlined workflow for sample collection and analysis as well as downstream data analysis using R. We detected phenotypic differences in the CSF cells of MS patients compared to those with the other neurological conditions. Our findings underscore the importance of a detailed characterization of immune cell profiling in CSF, which has high potential use as a diagnostic tool and as a measure of disease activity, enhancing our comprehension of the factors contributing to diseases heterogeneity such as AD and MS.