Objective: The aim of this study was to investigate the sensitivity of chemotherapeutic agents 5-FU, cisplatin (DDP) or TAXOL on colon cancer cell line SW480 with different methods, to find out the best examine time period for further study of chemotherapeutic sensitivities. Methods: The SW480 cell was treated with 5-FU (200 μg/mL), DDP(150 μg/mL) or TAXOL (50 μg/mL) respectively for 4 h, 8 h or 12 h. MTT assay was used to examine the cell survival rate, Annexin V/PI assay was used to analysis the apoptosis rate, Western Blot assay was applied to examine the expression of apoptotic protein. Results: (1) Results of MTT assay showed that the survival rate of SW480 cells at 4h, 8 h or 12 h was: 5-FU (200 μg/mL) 96.0% ± 8.2%, 85.4% ± 7.8%, 74.4% ± 10.2% (P < 0.05); DDP (150 μg/mL) 99.0% ± 6.4%, 88.7% ± 4.7% (P < 0.05), 46.9% ± 2.6% (P < 0.01); TAXOL (50 μg/mL) 51.5% ± 4.2% (P < 0.01), 31.9% ± 3.1%, 17.6% ± 2.3%, or blank group 97.2% ± 5.8%, 98.7% ± 7.2%, 97.5% ± 7.5% respectively. (2) The apoptosis rate of cancer cells at 4 h, 8 h or 12 h was: control group: 3.4% ± 0.2%, 6.2% ± 0.4%, 7.0% ± 0.5%; 5-FU (200 μg/mL) 4.0% ± 0.3%, 4.8% ± 0.4%, 7.7% ± 0.7%; DDP (150 μg/mL) 8.5% ± 0.9%, 18.6% ± 1.6% (P < 0.05), 67.0% ± 6.2% (P < 0.01); or TAXOL (50 μg/mL) 32.0% ± 5.2% (P < 0.01), 76.6% ± 8.5%, 94.0% ± 8.2%, respectively. (3) Western Blot assay showed that the expression of apoptosis associated protein. PARP, X-linked inhibitor of apoptasis (XIAP), Caspase-9 and Bcl-xL were changed. Conclusion: The sensitivity of chemotherapy could be assessed by MTT assay, Annexin V/PI assay and Western Blot. The best examine time of the three chemotherapuc agents was 5-FU (200 μg/mL): > 12 h, DDP(150 μg/mL): 8-12h, or TAXOL (50 μg/mL): < 4 h. © 2011 Springer-Verlag.