Abstract: Objective: To compare the value of self-established three-dimensional and three-step MTT assay (3D3S-MTT assay),MTT assay and Sulforhodamine B (SRB) assay for tumor chemosensitivity testing. Methods: Hep-G2,T-24and SKOV3 cell lines were exposed to Adriamycin and Mitomycin C for 3days with different further culture duration and then carried out the above three methods.For MTT assay and SRB assay the equation to calculate the percentage of tumour growth inhibition (%T GI) is:%T GI=1-A_t/A_c×100%.According to the self-established mathematical model,the equation for3D3S-MTT assay is:%T GI=1-lnA_max-ln(A_max-A_t)/lnA_max-ln(A_max-A_c)^1/b ×100%. Results: S-shaped drug-response curves were showed in all the above three methods.Comparison of the results of %T GI and IC₅₀ show that%T GI predicted by MTT assay is lower than that by SRB assay or3D3S-MTT assay,and IC₅₀ predicted by MTT assay is in average 3 to 5 folds of that by SRB assay or3D3S-MTT assay.%T GI and IC₅₀ predicted by3D3S-MTT assay is approximately the same value as predicted by SRB assay. Conclusion: 3D3S-MTT assay is applicable for in vitro tumor chemosensitivity testing,which is a successful solution to the3 to 5folds underestimation of chemosensitivity of MTT assay compared with SRB assay.