Hookworm is one of the most prevalent neglected tropical diseases in the world, infecting nearly 1.5 billion people worldwide. Severe cases of hookworm can lead to severe anemia, which can cause developmental complications in young children. The current method of hookworm diagnosis involves egg detection from stool samples via a Kato–Katz smear under a microscope, which is neither efficient nor sensitive. Hookworm diagnosis involving amplification of species-specific cell-free DNA from urine samples is feasible, as hookworm DNA can be filtered out of urine. The goal is to demonstrate this feasibility so that working with stools can be avoided in the future. Different concentrations of ITS-2 and repeat primers for the amplification were tested, and 250 nM consistently demonstrated the best amplification. Six urine samples of approximately 40-50 mL were collected from Marquette University volunteers. Nine different serial dilutions of genomic DNA (gDNA) of Necator americanus and Ancylostoma duodenale, demonstrating high, medium, and low concentrations, were prepared (3 different concentrations x 3 serial dilutions of each concentration = a total of 9 combinations). The nine combinations for each species were then filtered using Whatman grade 3 filter paper, air dried, and packed in a Ziploc bag with desiccant. Then, DNA was extracted using the QIAMP Blood Mini Kit and Nanodrop for concentration. These extracted DNAs are now being amplified using species-specific primers for both the ITS-2 gene and repeat fragments, including negative and positive controls, and will be visualized using gel electrophoresis. This is a novel assay that will reduce the need for using stools in the future, leading to a faster, more sensitive, and more specific assay that can help facilitate targeted mass drug administration.