A rapid glasshouse-based bioassay method to screen large numbers of cotton plants for responses to Fusarium oxysporum f. sp. vasinfectum (Fov) was developed. Different Fov inoculum concentrations and methods of inoculation were assessed using resistant and susceptible cotton cultivars. Cotton seeds were planted directly into Fov-inoculated soil. Studies of seed germination, seedling establishment, seedling mortality and fusarium wilt symptoms (i.e. stunting, foliar symptoms and vascular browning) were performed to optimize the bioassay parameters. Growing seedlings in Fov-inoculated soils at 5 × 104 or 1 × 105 CFU g−1 soil, in individual seedling tubes with 12 h at 28–30°C and 12 h at 15–18°C, gave consistent results when assessing Fov disease responses 6 weeks after inoculation. When fusarium wilt resistance ranks (FWRRs) and vascular browning index (VBI) means of 18 Australian and other cotton cultivars from the Fov glasshouse bioassay were compared against their fusarium field performance ranks (F-ranks), assessed on adult plants for cotton cultivar release, Pearson’s correlation was highly significant for both comparisons. The level of congruence between field and glasshouse data indicated that this protocol should be an effective tool for large-scale screening for Fov-resistance responses in diverse germplasm and breeding populations and for advancing genetic research to develop molecular markers for Fov resistance in cotton.