Epigenetic modifications of DNA, including methylation, hydroxymethylation, formylation, and carboxylation of cytosines, are proposed to function in gene regulation during reproduction and development. Changes in cytosine methylation are associated with a range of diseases, such as cancer. Immunofluorescence uses specific antibodies to quantitatively detect the global amount of cytosine modifications by fluorescence microscopy. The most critical stage of immunofluorescence is the antigen retrieval to remove the protein content around the DNA, allowing specific antibodies to bind to DNA epitopes. Acid treatments have commonly been used for antigen retrieval. Previously, trypsin was added after acid in the protocol, which increased the amount of detectable DNA methylation. In this study, the protocol was further enhanced by the addition of pepsin, which is able to target charged hydrophobic amino acids in proteins, unlike trypsin, which breaks positive hydrophilic amino acids. The global levels of cytosine modifications in CF-1, HeLa, and AR42J cells were compared using this protocol. In all cells, the sequential treatment of trypsin and pepsin increased the specificity of the staining. With the synergistic effect of the two enzymes, it is possible to target different protein groups packaging DNA molecules and removing them effectively. The findings suggest that this revised protocol can be conveniently used for each cytosine modification in the cells examined, and should be optimized for other cells. These new antigen retrieval conditions may more accurately detect the changes in cytosine modifications during development and in diseases.