BACKGROUND: Bioassays evaluating entomopathogenic fungi (EPF) isolates for effective microbial control of whitefly are a fundamental part of the screening process for bioprotectants, but development of repeatable, robust bioassays is not straightforward. Currently, there is no readily available standardised method to test the efficacy of EPF on whitefly. Here, we describe the calibration and use of a spray tower to deliver a standardised protocol to assess EPF activity; the method was validated using 18 EPF from four genera in tests against greenhouse whitefly, Trialeurodes vaporariorum (Westwood).RESULTS: At 138 kPa, the sprayer delivered 0.062 mL mm −2 (620 L ha −1 ) and an even deposition of spray across the central 1590 mm 2 of the spray area. Average conidial deposition for all EPF was 252 conidia mm −2 and equivalent to 2.5 × 10 12 conidia ha −1 at an application concentration of 1 × 10 7 conidia mL −1 . Conidial deposition of a test Beauveria bassiana suspension increased with increasing application concentration. Egg laying by T. vaporariorum adults was restricted to 177 mm 2 using clip cages specifically designed to ensure that third-instar T. vaporariorum received a uniform spray coverage. Nymphs occupied 373 ± 5 mm 2 of the leaf after migrating during the first instar. Average T. vaporariorum mortality totaled 8-89% 14 days after application of 1 × 10 7 conidia mL −1 of each EPF isolate. CONCLUSION: Combining the calibrated sprayer and bioassay method provides a reliable, standardised approach to test the virulence of EPF against whitefly nymphs. This laboratory-based assay is affordable, replicable and allows the user to alter the dose of conidia applied to the target.