PURPOSE Both chronic hepatitis C (HCV) and B virus (HBV) infections are generally asymptomatic, and many remain undetected or are diagnosed at a late stage. Studies that evaluate best practice hepatitis testing strategies are needed to better detect this hidden population. METHODS In this prospective cohort study, we aimed to determine the diagnostic yield (test uptake and rate of positive test results) of a combined public health and primary care birth cohort testing strategy in detecting hidden cases of HCV and HBV infections. We invited all patients aged between 40 and 70 years (n = 6,743) registered with 11 family practices serving 2 higher prevalence areas, or hotspots (ie, estimated HCV prevalence of 1%; national estimated prevalence is 0.1-0.4%), in the south of the Netherlands. RESULTS Test uptake was 50.9% (n = 3,434 patients). No active or chronic HCV infection was detected: 0.00% (95% CI, 0.00%-0.11%). Positive test rates were 0.20% (95% CI, 0.08%-0.42%) for anti-HCV (n = 7), 0.26% (95% CI, 0.12%-0.50%) for hepatitis B surface antigen (n = 9), and 4.14% (95% CI, 3.49%-4.86%) for antihepatitis B core (n = 142). CONCLUSIONS This best practice testing strategy was effective in achieving a high test uptake. It completely failed, however, to detect hidden chronic HCV infections and is not recommended for countries with a low prevalence of the disease.