Cells cultured in three dimensional (3D) scaffolds as opposed to traditional two-dimensional (2D) substrates have been considered more physiologically relevant based on their superior ability to emulate the in vivo environment. Combined with stem cell technology, 3D cell cultures can provide a promising alternative for use in cell-based assays or biosensors in pre-clinical drug discovery studies. The number of research publications about 3D cell culture in vitro models is increasing drastically in the recent decade; however, the adoption of 3D cell culture in industry is slow. The possible reasons include 1) lack of concrete evidence for in vivo emulation, 2) absence of measure to ascertain three dimensionality of the culture, and 3) high cost of commercial systems in a climate of great need to reduce cost of screening per compound. In this study, we aimed at advancing 3D culture application in neuronal cell based assays for pre-clinical drug discovery by attempting to solve the above three problems. First we established the in vivo emulating property of 3D cultured neuronal cells by comparing with freshly dissected tissue as in vivo surrogate. Intracellular calcium transient in response to high K+ depolarization was used as a convenient index to access the physiological relevance of 3D cultured cells. Membrane protein caveolin-1, alpha subunit of L-type VGCC and cytoskeletal protein F-actin were also compared in this study to shed light on the mechanism behind the phenomenon. The second step is to identify potential three-dimensionality biomarkers for advancing 3D cell culture technology. With this goal in mind, transcriptomic expression comparison among neural progenitor cells cultured on 2D substrates, in 3D porous polystyrene scaffolds, and as 3D neurospheres (in vivo surrogate) was conducted. Up-regulation of cytokines as a group in 3D and neurospheres was observed. The commonly up-regulated cytokines were identified and discussed in depth for their promising role as 3D biomarkers.Last, automation and robotic techniques were utilized to generate a HTS compatible 3D cell culture platform by chemically welding polystyrene scaffolds into standard 2D polystyrene 96-well plates. Automation can minimize the variability of scaffold properties and increase the Z-factor of the produced 96-well plates. The fabricated 3D cell culture plates were compared with several commercially available 3D cell culture platforms to confirm the suitability in pre-clinical drug discovery applications.