The global strategy to eradicate Hepatitis B (HBV), Hepatitis C (HCV), and HIV by 2030 is critical due to their impact and challenges to healthcare systems. HCV is curable, but HBV and HIV are only suppressible, with a vaccine available solely for HBV. Innovative diagnostic methods are needed, especially for high-risk populations like people who inject drugs (PWID). This study validates a dried blood spot (DBS) nucleic acid amplification test (NAAT) using the Hologic Panther system for detecting HBV, HCV, and HIV. The method was used to screen among PWID in the Capital Region of Denmark. The DBS method demonstrated high sensitivity, with a 95 % limit of detection (LoD) of 2711 IU/mL for HBV, 525 IU/mL for HCV, and 4022 copies/mL for HIV. Screening of 83 PWID in Denmark revealed a 13 % prevalence of active HCV infection, offering significant benefits in settings where traditional venous access is difficult.