Limited efficacy of chemotherapeutic drugs against solid tumors has been attributed to poor drug penetration into tumor tissues. Multicellular layer (MCL) cultures recapitulate barriers to drug penetration and distribution and have been used successfully in the production of clinically relevant data. In the present study, we evaluated the characteristics of paclitaxel (PTX) and 5-fluorouracil (5-FU) penetration and their effects on tissue penetration using MCLs of human colorectal cancer cells (DLD-1 and HT-29) grown in Transwell inserts. Drug concentration in conditioned media after MCL penetration was estimated using % survival of cells exposed to the conditioned media, and the penetration rate was calculated as % drug concentration relative to the expected concentration after penetration of cell-free MCLs. PTX showed limited penetration in both MCLs in contrast to the full penetration seen by 5-FU. The penetration rate measured after 24 h by cytotoxicity of the conditioned media was 40 and 38% in DLD-1 (20 μM) and HT-29 MCLs (1 μM), respectively, at which concentration the conditioned media produced 50% growth inhibition in monolayers. The penetration profile obtained using [14C]-paclitaxel also showed slow and limited penetration with concentration- and cell line-dependency. In HT-29 MCL, full penetration of PTX was obtained at 10 μM after 48 h, whereas only 80% was obtained at 1 μM. In DLD-1 MCLs, penetration of PTX was minimal, especially at 1 μM, showing penetration rates as low as 10 and 20% after 24 and 96 h, respectively. When PTX and 5-FU were allowed to penetrate in sequential combination, no effect on the penetration rate was observed. Overall, our results demonstrated limited penetration of PTX in human colorectal cancer MCLs along with concentration-, time-, and cell line-dependency. Assessment of penetration using cytotoxicity of the conditioned media used in the present study may be useful in early stage screening of anticancer agents for their potential in tissue penetration.