Infertility is an issue for approximately 12% of couples attempting to have a child. Of this group, 50% of the cases are due to male factor infertility. There are many reasons for decreased fecundity in men, but there remains 10% to 15% of infertile men that are diagnosed with the most severe form of infertility, non-obstructive azoospermia (NOA). A diagnosis of NOA implies the lack of sperm cells in the ejaculate with no physiological reason. The current diagnostic test and treatment consist of microscopic examination of seminal fluid and a biopsy to extract any viable sperm from the testis. This treatment is known to be problematic because of the destructive nature of surgery as well as expense. A non-invasive diagnostic test that could identify the presence of sperm in the testis at the beginning of fertility treatment would inform the patient and the physician about the functionality of the testis and thus lead to more informed decisions about treatment and potentially a decrease in cost. The ability to identify the tissue source of DNA present in the reproductive tract could facilitate a fertility diagnostic tool. Tissue specific epigenetic mechanisms are known to play a role in an organism's development. The identification of an epigenetic signature unique to sperm DNA would allow for the identification of sperm DNA in a heterologous mixture. Our lab has been able to identify a methylation signature that can consistently differentiate between sperm DNA and somatic DNA. We compared the sperm DNA signature with that of blood and testicular tissue and found that there was no overlap in epigenetic markers. To create an assay that could evaluate the presence of sperm DNA we used an Oxford Nanopore next-generation sequencing platform. Sequencing bisulfite converted DNA; we were able to retrieve the methylation status at locations of interest. A bioinformatic tool was created to analyze the thousands of reads obtained and analyze the individual methylation points within single molecules of DNA. To create a more accessible fertility test, we used the sperm DNA analysis tool to evaluate seminal cell-free DNA (cfDNA). The presence of sperm cfDNA in a patient's seminal fluid may indicate that there is sperm somewhere in the male reproductive tract even if the cells are not intact. A clinician could use this information to better advise the patient about treatment and potentially decrease cost of care.