Photochemical treatment (PCT) of platelet concentrates, using amotosalen HCl and UVA-light, inactivates pathogens by forming adducts between amotosalen and nucleic acids. The impact of the photochemical treatment on pathogens and leukocytes has been studied extensively. Yet little is known about the effect of PCT on nucleic acids in platelets. Platelets contain viable mitochondria and mitochondrial DNA (mtDNA) and this study aimed at evaluating the amotosalen modifications on platelet mtDNA. We applied two independent but complementary molecular assays to investigate qualitative as well as quantitative aspects of the psoralen-mediated DNA modifications in platelet mtDNA. The amotosalen-DNA modification density was measured using ¹⁴C-labeled amotosalen. Amotosalen (150 µM) yielded 4.0 ± 1.2 psoralen adducts per 1000 bp in mtDNA after irradiation with 3 J/cm² UVA. Furthermore, we tested if the PCT-induced DNA modifications could be detected by a PCR assay. On the basis of PCR inhibition due to amotosalen–DNA adducts, mtDNA-specific PCR assays were developed and tested for their specificity and sensitivity. Our data revealed that mtDNA in platelets is substantially modified by PCT and that these modifications can be documented by a PCR inhibition system.