Three-dimensional (3D) multicellular spheroids have been identified as a suitable model of solid tumours for drug screening and cancer research [1]. However, creating and monitoring a large number of spheroids using conventional methods remains labour-intensive. This work reports a novel double-layer microfluidic device for the generation and culture of 3D multicellular spheroids and its use with biopsy samples. The system enables drug concentration-response curves to be obtained from a limited amount of sample material by applying a drug concentration gradient using an integrated passive microchannel network