Cell-based assays can be applied to evaluate the efficiency of anti-cancer drugs but the conventional approaches are mostly based on two-dimensional cell culture which is not able to recapitulate the tumour specificity. Here we developed a method to culture millimetre size tumour spheroids that is useful for anticancer drug studies. Agarose multi wells were obtained by casting on polymethylsiloxane (PDMS) mould, which were then used for culture of U87-MG human glioblastoma. As expected, large size tumour spheroids could be generated after 24 h incubation. Comparing to the multi-well systems made of PDMS or polyethylene glycol diacrylate (PEGDA), agarose multi wells are clearly advantageous due to the hydrophobic surface and the high permeability of agarose. After culture for 10 days, the tumour spheroids in agarose wells stopped to grow and the further increase of the cell seeding density had no effect on the final size of the spheroids. To study the anticancer drug effect, combretastatin A-4 (CA4) was added on day 2 or day 4, showing clear effects on the tumour spheroids and cell viability. More importantly, the live/dead cell staining images suggested that an earlier drug treatment was more efficient to prohibit the tumour spheroid growth.