Microplastics and nanoplastics are increasingly detected in food and drinking water, but their impact on mitochondrial DNA (mtDNA) integrity remains unclear. Specifically, how ingested nanoplastics exposure increases mtDNA strand breaks in Drosophila melanogaster larvae is yet to be determined. Larvae are raised on food containing defined nanoplastic concentrations to model particle accumulation and cellular stress following nanoplastic ingestion. Larval digestional tracks are dissected out, and DNA is extracted. mtDNA damage is quantified from extracted DNA using qPCR, where reduced amplification of a long mtDNA target compared to a short amplified target indicates increased lesioning. Ultimately, this study provides an assessment of mitochondrial genotoxicity following nanoplastic ingestion, helping to define mitochondrial genome damage as a potential contributor to nanoplastic-associated cellular dysfunction.