AbstractBackgroundA challenge frequently encountered in neuroepigenomic research, especially in the context of neurodegenerative diseases such as Alzheimer’s disease, is the difficulty of measuring epigenetic patterns in heterogenous bulk tissue samples, as these are known to cause noise during profiling. A viable strategy to overcome this challenge is to analyze epigenetic marks in single neurons that have been isolated using laser capture microdissection (LCM).MethodIn this study, we developed a new approach for targeted DNA methylation profiling of individual genes that combines the use of LCM and limiting dilution bisulfite pyrosequencing (LDBSP). By utilizing this method, we were able to determine the cytosine‐phosphate‐guanine (CpG) methylation rates of single alleles obtained from 50 neurons, which were isolated from unfixed post‐mortem brain tissue.ResultWe demonstrate how the methylation status of multiple genes such as RHBDF2, OXT, TNXB, DNAJB13, PGLYRP1, C3, and LMX1B, can be quantified simultaneously using the method described above. In addition, we provide an adapted data analysis pipeline for LDBSP, which accounts for and corrects methylation rates obtained from multi‐allele reactions. Furthermore, we show that the efficiency of LDBSP on DNA derived from LCM‐isolated neurons is comparable to the efficiency in previous studies using other cell types.ConclusionThis method allows for a more precise determination of the DNA methylation status of each targeted gene in the neuronal cell samples under investigation, thereby increasing the validity of the technique.