A room temperature biochemical assay, based on centrifugal removal of tubulin polymer, was developed to permit ready detection of paclitaxel analogs more active than the parent compound and to permit reliable quantification of differences in activity relative to paclitaxel in terms of drug concentration. The assay was validated by comparing paclitaxel to two compounds (docetaxel and 2-debenzoyl-2-meta-azidobenzoylpaclitaxel) known to be more active under multiple reaction conditions. The assay was designed to yield a relatively high EC50 (23 microM) for paclitaxel. This was possible because paclitaxel only weakly induced tubulin assembly at room temperature in 0.4 M glutamate without exogenous GTP. Under these same reaction conditions 50% assembly occurred with 4.7 microM 2-debenzoyl-2-meta-azidobenzoylpaclitaxel and 11 microM docetaxel. These biochemical EC50 values were in agreement with the relative cytotoxicity of the three compounds for human Burkitt lymphoma CA46 cells (IC50 values for paclitaxel, docetaxel, and 2-debenzoyl-2-meta-azidobenzoylpaclitaxel were 40, 10, and 3 nM, respectively).