Three-dimensional (3D) spheroid models offer a more physiologically relevant and complex environment compared to traditional two-dimensional cultures, making them a promising tool for studying tumor biology and drug response. However, these models often face challenges in real-time monitoring of drug diffusion, penetration, and target engagement, limiting their predictive power for in vivo and clinical outcomes. This study introduces a novel approach for real-time tracking of drug permeability using small molecule drugs with GFP/RFP-disrupting properties that correlate with their efficacy. We developed a reproducible 3D spheroid model with various cancer cell lines expressing GFP/RFP for efficient drug screening. Through screening over 20 FDA-approved enzyme inhibitors, we identified three covalent kinase inhibitors-osimertinib, afatinib, and neratinib-that irreversibly disrupt GFP and RFP fluorescence. Our results reveal distinct drug diffusion and penetration profiles within GFP/RFP-expressing spheroids, varying with drug concentration and formulation, and correlating with clinical volume of distribution (Vd) values. Additionally, we demonstrate that our approach is useful for evaluating different drug formulations as well as screening penetration enhancers for solid tumors. These findings offer a valuable 3D model for studying kinetics of drug permeability and efficacy in tumor-like environments, with potential implications for drug delivery research and formulation development.