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The use of alternative techniques to reduce the number of animals used in anticancer research is an issue of current interest. The aim of this study was to validate the use of a simple and efficient alternative tool for the assessment of the potential of novel anti‑proliferative agents. A set of 20 compounds with various mechanisms were tested in the Triticum aestivum root elongation assay, using aminophylline as negative control. Hierarchical cluster analyses were performed using the furthest neighbor method based on Euclidean distance measure, and the compounds were statistically analyzed in reference to their anti‑proliferative pattern registered in the NCI60 human tumor cell line anticancer drug screen. A correlation between the Triticum test results and the NCI60 anti‑proliferative profile was made for a number of human cells that we defined as the Triticum cell panel. Linear equations were computed that can be used to transform the inhibitory effect measured in any future Triticum assay in order to predict the effect on particular human cells. Of the tested anti‑proliferative agents, methotrexate, colchicine, cantharidin, cisplatin and verapamil produced a growth inhibition over 50%. On the whole, the findings of this study suggest that the Triticum test can be used to detect several types of anti‑proliferative mechanisms, particularly those targeting tubulin, rendering it a useful tool with which to identify novel mitotic spindle inhibitors.