The introduction of blood donor screening by virus nucleic acid amplification technology (NAT) in the mid to late 1990s was driven by the so-called AIDS and hepatitis C virus (HCV) epidemic, with thousands of recipients of infected blood products and components. Plasma fractionators were the first to introduce NAT testing besides pathogen reduction procedures, to reduce the virus transmission risk through their products. To achieve a similar safety standard, NAT was then also introduced for labile blood components. German transfusion centres were the first to start in-house NAT testing of their donations in pools of up to 96 samples for HCV, hepatitis B virus (HBV), and human immunodeficiency virus-1 (HIV-1). Years later the diagnostics industry provided commercial HCV and HIV-1 and later HBV NAT tests on automated platforms. NAT tests for HIV-2, hepatitis A virus, and Parvovirus B19 followed, again driven by transfusion centres with their in-house tests. When severe acute respiratory syndrome corona virus (SARS-CoV) and West Nile Virus emerged it was the NAT that enabled the manufacturers and transfusion centres to instantly introduce sensitive and specific screening tests. Subsequent automation including sample preparation has significantly reduced the costs and complexity of the procedure and made it affordable to middle income countries as well. Currently more than 60 million donations per year are NAT tested worldwide and the remaining residual risk of virus transmission by blood components and products could be reduced to almost zero. Automation rendered possible the reduction of pool size in conjunction with increased throughput and sensitivity. Thus, antibody and antigen testing may be dispensable in the long run, particularly in the combination of NAT testing with pathogen reduction. There are new technologies on the horizon like digital droplet PCR, next-generation sequencing, lab-on-a-chip, and digital antigen assays, which are comparably sensitive. However, each of these has limitations, either in throughput, costs, automation, time to result, specificity, or the need for NAT as an integral part of the technology. Thus, NAT is still the shortest and most efficient means to the result. Donor screening NAT also contributed significantly to our knowledge on how fast viruses replicate, and on the respective diagnostic window. In conjunction with animal and patient studies, we have learned more about the minimal infectious dose and the epidemics in the donor population.