A rapid method for determining the response of cereals to glyphosate is described. This method detects the differential responses of plants in 4 d, allowing for the rapid selection of glyphosate-tolerance response. Two types of tests determined the efficacy of this rapid method: differential response to different dosages of herbicide in a coleoptile growth test and in sprayed plants. In seedling assay, barley cultivars showed a higher level of tolerance to glyphosate (the dose that causes 50% of the total effect [I50] = 0.066 and I50 = 0.060 mM) than wheat cultivars (I50 = 0.018 and I50 = 0.014 mM). The response in seedling assay is well correlated (r2 = 0.95 and r2 = 0.98) with the response in plant-sprayed assays. The method was employed to verify the tolerance level of a sensitive barley cultivar and the four tolerant and sensitive mutant lines derived from it.