DNA Methylation, 5meC, is an epigenetic modification that acts as an important regulator of genomic stability and gene expressivity. Genome-wide changes in methylation have been associated with lineage-specific changes in gene expression profiles during development and in some cell-based pathologies, including oncogenesis. Cost-effective and rapid platforms for the detection of changes in the global levels of methylation are of value for the investigation of the processes that regulate methylation. Flow cytometry allows rapid and quantitative analysis of epitopes within a large number of cells. We have recently optimised the conditions required for valid detection of 5meC by immunofluorescence microscopy. These studies showed that immunological detection of 5meC requires the sequential denaturation of chromatin by a brief period of acidification followed by a partial tryptic digestion step. We have assessed the reliability of flow cytometry for the detection of changes in 5meC when coupled with this optimised epitope retrieval strategy. This study provides support for the use of high throughput screening of 5meC by flow cytometry for the analysis of the epigenetic regulation of important cell transitions.