Both hepatitis C virus (HCV) infection and human immunodeficiency virus (HIV) infection areunderdiagnosed, particularly in low-income countries and in difficult-to-access populations. Our aim was to develop and evaluate a methodology for the detection of HCV and HIV infection based on capillary dry blood spot (DBS) samples taken under real-world conditions. We carried out a crosssectional study of 139 individuals (31 healthy controls, 68 HCV-monoinfected patients, and 40 HCV/ HIV-coinfected patients). ELISA was used for anti-HCV and anti-HIV antibody detection; and SYBR Green RT-PCR was used for HCV-RNA detection. The HIV serological analysis revealed 100% sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The HCV serological analysis revealed a sensitivity of 92.6%, specificity of 100%, PPV of 100%, and NPV of 79.5%. Finally, the HCV-RNA detection test revealed a detection limit of 5 copies/µl with an efficiency of 100% and sensitivity of 99.1%, specificity of 100%, PPV of 100%, and NPV of 96.9%. In conclusion, our methodology was able to detect both HCV infection and HIV infection from the same DBS sample with good diagnostic performance. Screening for HCV and HIV using DBS might be a key strategy in the implementation of national programs for the control of both infections.Hepatitis C virus (HCV) infection is a major health problem worldwide. Approximately 71 million persons live with HCV infection, and around 80% are undiagnosed 1 . Infection by human immunodeficiency virus (HIV) continues to be a major public health issue, with a global prevalence of 0.8% among adults, of whom around 30% are undiagnosed 2 . Both HIV and HCV share routes of transmission, and their diagnosis is a priority in public health strategies throughout the world 1,2 . On the one hand, HIV infection is diagnosed increasingly frequently in resource-limited settings through implementation of HIV testing programs; however, HIV testing is still not reaching some communities (eg, people who inject drugs [PWID], sex workers, homeless persons), resulting in late presentation 3 . On the other hand, diagnosis of HCV remains problematic for many low-income countries and difficult-to-access populations in developed countries 4 , where most individuals remain unaware of their HCV status until advanced stages of the disease 5-7 .Traditionally, diagnosis of HIV and HCV infection has been based on the detection of antibodies, antigens, and the viral genome in blood samples obtained by routine venous blood collection 5,8 . However, the use of capillary whole blood in dried blood spot (DBS) specimens may be an excellent alternative in both serological and nucleic acid testing (NAT) assays for the screening of HIV and HCV infection, since these approaches are less invasive, samples can be stored and transported without refrigeration, and highly trained personnel are not necessary 5,9,10 . Therefore, DBS may facilitate diagnosis of HIV and HCV infection in settings where access to screening is difficult. In fa...