Three experiments were conducted to develop a bioassay method for assessing the bioavailability of prosulfocarb, pyroxasulfone and trifluralin in both crop residue and soil. In preliminary experiments, Italian ryegrass (Lolium multiflorum Lam.), cucumber (Cucumis sativus L.) and beetroot (Beta vulgaris L.) were tested as bioassay plant species for the three pre-emergent herbicides. Four growth parameters (shoot length, root length, fresh weight and dry weight) were measured for all plant species. Shoot-length inhibition was identified as the most responsive to the herbicide application rates. Italian ryegrass was the most sensitive species to all tested herbicides, whereas beetroot and cucumber had lower and similar sensitivity to shoot inhibition for the three herbicides. The bioassay species performed similarly in wheat and canola residues collected a few days after harvest. In bioassay calibration experiments, dose–response curves were developed for prosulfocarb, pyroxasulfone and trifluralin in a sandy loam soil typical of the grain belt of Western Australia and with wheat residue. The developed bioassay uses ryegrass shoot inhibition for relatively low suspected concentrations of herbicide, and cucumber shoot inhibition for higher rates. The bioassay was validated by spraying the three herbicides separately onto wheat residue and soil and comparing the concentrations derived from chemical analysis with those from the bioassay. All of the linear correlations between concentrations derived from chemical analyses and the bioassays were highly significant. These results indicate that the bioassay calibration curves are suitable for estimating herbicide concentrations in crop residue collected soon after harvest and a sandy-loam soil, low in organic matter.