SUMMARY. Thirty-one outbreaks of Marek's disease (MD) were reported in the Netherlands and retrospectively analyzed. The outbreaks occurred mostly in vaccinated commercial layer and a few breeder flocks of several breeds; however, the cause of the outbreaks could not be established. Therefore, in a prospective study, the occurrence of true vaccine failures was assessed on five hatcheries. The plaque-forming units (PFU) of MD vaccine per chicken dose were determined through in vitro assays on vaccine ampoules (2 to 5 per hatchery) and samples of reconstituted vaccine (approximately 22 per hatchery). All forty reconstituted vaccine samples of hatcheries 1 and 4 showed PFU doses <10³. In hatchery 4, 14 samples showed extreme low PFU (≤10 PFU). In hatcheries 2, 3, and 5, the numbers of MD vaccine suspensions with a titer ≥10³ PFU, which is the standard required, were 1 (5%), 17 (77%), and 3 (14%), respectively. Some vaccine ampoules showed <10³ PFU per chicken dose. This study shows the usefulness to assess the PFU per chicken dose of reconstituted MD vaccine and vaccine ampoules to unravel true vaccine failures, which could result in disease outbreaks in the field.