A new microfluidic cell culture device compatible with real-time nuclear magnetic resonance (NMR) is presented here. The intended application is the long-term monitoring of 3D cell cultures by several techniques. The system has been designed to fit inside commercially available NMR equipment to obtain maximum readout resolution when working with small samples. Moreover, the microfluidic device integrates a fibre-optic-based sensor to monitor parameters such as oxygen, pH, or temperature during NMR monitoring, and it also allows the use of optical microscopy techniques such as confocal fluorescence microscopy. This manuscript reports the initial trials culturing neurospheres inside the microchamber of this device and the preliminary images and spatially localised spectra obtained by NMR. The images show the presence of a necrotic area in the interior of the neurospheres, as is frequently observed in histological preparations; this phenomenon appears whenever the distance between the cells and fresh nutrients impairs the diffusion of oxygen. Moreover, the spectra acquired in a volume of 8 nl inside the neurosphere show an accumulation of lactate and lipids, which are indicative of anoxic conditions. Additionally, a basis for general temperature control and monitoring and a graphical control software have been developed and are also described. The complete platform will allow biomedical assays of therapeutic agents to be performed in the early phases of therapeutic development. Thus, small quantities of drugs or advanced nanodevices may be studied long-term under simulated living conditions that mimic the flow and distribution of nutrients.