We present the characterisation and validation of multiplexed 4-terminal (4T) impedance measurements as a method for sensing the spatial location of cell aggregates within large three-dimensional (3D) gelatin scaffolds. The measurements were performed using an array of four rectangular chambers, each having eight platinum needle electrodes for parallel analysis. The electrode positions for current injection and voltage measurements were optimised by means of finite element simulations to maximise the sensitivity field distribution and spatial resolution. Eight different 4T combinations were experimentally tested in terms of the spatial sensitivity. The simulated sensitivity fields were validated using objects (phantoms) with different conductivity and size placed in different positions inside the chamber. This provided the detection limit (volume sensitivity) of 16.5%, i.e. the smallest detectable volume with respect to the size of the measurement chamber. Furthermore, the possibility for quick single frequency analysis was demonstrated by finding a common frequency of 250 kHz for all the presented electrode combinations. As final proof of concept, a high density of human hepatoblastoma (HepG2) cells were encapsulated in gelatin to form artificial 3D cell constructs and detected when placed in different positions inside large gelatin scaffolds. Taken together, these results open new perspectives for impedance-based sensing technologies for non-invasive monitoring in tissue engineering applications providing spatial information of constructs within biologically relevant 3D environments.