An experiment was conducted to develop and optimize a chicken bioassay to detect the presence of infective viral pathogens in poultry litter from a variety of sources. The experiment also aimed to determine the effect of type of chickens and age of exposure to litter on the level of viral infectivity. The bioassay detected chicken anaemia virus, infectious bursal disease virus and fowl adenovirus from chicken litters. SPF chickens showed higher sensitivity than commercial broiler chickens without any effect of age of exposure, however, the assay was more sensitive in broiler chickens when exposed at day 8. We conclude that the bioassay based on exposure of day-old SPF chickens is a viable assay of viral infectivity of poultry litter.