The development of novel cellular models that can replace animals in preclinical trials of drug candidates is one of the major goals of cell engineering. Current in vitro screening methods hardly correspond with the in vivo situation, whereas there is a lack of assays for more accurate cell culture models. Therefore, development of automated assays for 3D cell culture models is urgently required. In this work, we present a SpheroChip system: a microfluidic-based platform for long-term 3D cell culture and analysis. The system is compatible with commercially available microplate readers and provides continuous, in situ monitoring of tumour spheroids cultured on a chip. The microfluidic chip consists of cell culture microchambers and hemispherical microwells connected with a concentration gradient generator. HT-29 and Hep-G2 cells were successfully cultured as tumour spheroids in the SpheroChip, and metabolic activity of cells was monitored for up to two weeks by in situ fluorimetric measurements. Cellular response to an anticancer drug was observed using the SpheroChip. The experimental setup provided the unique possibility of observing dynamic changes in metabolic activity of one culture during sequencing days after drug dosage. According to this new approach, unknown phenomena of cellular response to the anticancer drug were observed, such as increase of metabolic activity shortly after drug dosage. Moreover, the influence of a second dose of a drug was evaluated. The SpheroChip system can be used by researchers working on drug screening, evaluation of anticancer procedures and chemoresistance phenomena.