R loops (DNA-RNA hybrid) are three-stranded nucleic acid structures that comprise of template DNA strand hybridized with the nascent RNA leaving the displaced non-template strand. Although a programmed R loop formation can serve as powerful regulators of gene expression, these structures can also turn into major sources of genomic instability and contribute to the development of diseases. Therefore, understanding how cells prevent the deleterious consequences of R loops yet allow R loop formation to participate in various physiological processes will help to understand how their homeostasis is maintained. Detection and quantitative measurements of R loops are critical that largely relied on S9.6 antibody. Immunofluorescence methods are frequently used to localize and quantify R loops in the cell but they require specialized tools for analysis and relatively expensive; therefore, they are not always useful for initial assessments of R loop accumulation. Here, we describe an improved slot blot protocol to detect and estimate R loops and show its sensitivity and specificity using the S9.6 antibody. Since specific factors protecting cells from harmful R loop accumulation are expanding, this protocol can be used to determine R loop accumulation in research and clinical settings.