<p>Most drug discovery studies use 2D cell cultures and animal models for screening new chemical entities (NCEs), which often leads to suboptimal results due to genetic variations, species differences, or lack of most physiological preclinical models. This is one of the most important reasons behind high rate of failure of drug candidate in the clinic, especially in oncology drug development projects. To address this issue, we developed a 3D pancreatic tumor spheroid model that better mimics the parental tumor architecture. We observed similar drug effects on cellular viability in both 2D cultures and 3D spheroids. However, cellular viability alone is insufficient to predict the translation of efficacy into clinical studies. A 3D multicellular tumor model is essential to comprehensively evaluate drug effects on the tumor microenvironment (TME), angiogenesis, and tumor biomarkers. Our model includes 3D monocellular and multicellular spheroids, which demonstrated a more relevant platform for potency evaluation. We used pancreatic ductal adenocarcinoma cells PANC-1 and PANC04.03 to conduct a comprehensive drug screening and assessed spheroid shrinkage and pre-vascularization. We also evaluated RT-qPCR analysis for gene expression of CSC markers (CD44, SOX2, KRT18), EMT markers (αSMA, vimentin) and the apoptotic marker (Annexin A1) under various conditions. Our findings highlighted the significant differences between 2D and 3D cultures, underscoring the importance of 3D multicellular models for predicting therapeutic markers and enabling comprehensive drug evaluation. In this study, MRTX1133 (a Phase I candidate of KRAS-G12D inhibitor) was used for testing our hypothesis. Treating the spheroids with MRTX1133 revealed enhanced drug response profiles compared to 2D cultures. This study underscores the critical importance of 3D multicellular model in preclinical drug screening and their potential to bridge the gap between in vitro studies and clinical outcomes.</p>