The in vitro cell culture models of human pluripotent stem cells (hPSC)-derived cardiomyocytes (CMs) have gained a predominant value in the field of drug discovery and is considered an attractive tool for cardiovascular disease modellings. However, despite several reports of different protocols for the hPSC-differentiation into CMs, the development of an efficient, controlled and reproducible 3D differentiation remains challenging. The main aim of this research study was to understand the changes in the gene expression as an impact of spatial orientation ofhPSC-derived CMs in 2D(two-dimensional) and 3D(three-dimensional) culture conditions and to identify the topologically important Hub and Hub-Bottleneck proteins using centrality measures to gain new knowledge for standardizing the pre-clinical models for the regeneration of CMs. The above-mentioned aim was achieved through an extensive bioinformatic analysis on the list of differentially expressed genes (DEGs) identified from RNA-sequencing (RNA-Seq). Functional annotation analysis of the DEGs from both 2D and 3D was performed using Cytoscape plug-in ClueGO. Followed by the topological analysis of the protein-protein interaction network (PPIN) using two centrality parameters; Degree and Betweeness in Cytoscape plug-in CenTiScaPe. The results obtained revealed that compared to 2D, DEGs in 3D are primarily associated with cell signalling suggesting the interaction between cells as an impact of the 3D microenvironment and topological analysis revealed 32 and 39 proteins as Hub and Hub-Bottleneck proteins, respectively in 3D indicating the possibility of utilizing those identified genes and their corresponding proteins as cardiac disease biomarkers in future by further research.