Leukocyte infiltration of the central nervous system (CNS) occurs under certain pathogenic conditions and most often results in severe disorders. Therefore, the isolation and analysis of such infiltrating cell populations is necessary for elucidating the underlying pathogenic mechanisms. Here we describe a simple and straightforward protocol for cell isolation from the inflamed CNS, which combines mechanical dissociation and enzymatic degradation of the tissue. Additionally, purification by Percoll gradient centrifugation provides a great yield of the infiltrating material. The isolated cells can be further used for downstream applications such as cell sorting, cellular or molecular analysis.