Purpose <p>Advances in drug discovery and early clinical development depends fundamentally on robust pre-clinical research and a comprehensive understanding of cancer biology. There is an international effort to reduce the use of animals in research and product development, with a focus on technical advances to build better in vitro models. Three-dimensional (3D) cell culture mimics the architecture of solid tumors and could be validated as an alternative method to animal use in research and development, promoting faster and more</p> Methods <p>A workflow of assays and decision points was established in the FIOCRUZ Technological Platform of Bioassays for antitumoral drugs at Farmanguinhos – FIOCRUZ. The flow with increased complexity starts with simple 2D assays, going through assays for determination of apoptosis and finally 3D assays for cytotoxicity and biomarker modulation. This drug discovery flow was challenged by two molecules received by our platform. As first step, compounds were tested in a single concentration using cell lines in the 2D model, with cytotoxicity assessment performed by the MTT assay. This stage is intended for early-phase projects (TRL 1 and 2), including the screening of new molecules, development of nanopreparations, and activity-guided synthesis or natural product fractionation. When at least 30% of cytotoxicity is observed, samples are forwarded for IC₅₀ determination and, if a good IC₅₀ is observed (below 10 µM) the selectivity index (SI) is determined and apoptosis analysis is performed. At this stage, the projects are already at more advanced maturity levels, covering lead compounds, enriched fractions, isolated natural products and optimized nanopreparations. Samples with good performance (IC₅₀ below 10 µM and SI above 3) are subsequently evaluated in 3D spheroid models using the forced suspension method, with the goal of complementing cytotoxicity data and then advancing to studies on biomarker expression. In this study, we have carried out assays on MCF-7 cell line.</p> Results <p>As expected, compounds RPT11M_10330 and RPT11M_10332 showed significant differences in IC₅₀ values between the 2D and 3D models. We observed that both compounds were cytotoxic and were equally effective as doxorubicin in the 2D model. The 3D model exhibited greater intrinsic resistance compared to the 2D model, as described in the literature; the Ultra-low attachment spheroid culture environment changed the tumor cellular responses to drugs as IC₅₀ values of these molecules were higher in the 3D model.</p> Conclusion <p>Each approach has its own strengths and limitations and should be considered both individually and in an integrated manner. Recent advances in preclinical screening tools, which more reliably predict clinical effects and adverse events of drug candidates, have begun a new era in drug development and screening. We suggest a combination of well-established 2D models with emerging techniques, such as 3D models, in drug research and development that may lead to robust preclinical reports, accelerating drug discovery projects.</p>