Physiologically relevant in vitro tumor models are crucial in any research setting from drug development to individualized treatment predictions. Patient-derived 3D cell culture models (PD3D®) are validated cancer models which recapitulate the biology of the donor tissue from genotype to phenotype. A multitude of PD3D® replicates can be generated and these are suitable for high-throughput-screening. However, the influence of media conditions on specific drug sensitivity profiles widely remains elusive and thus the predictive value of screenings is obscured. Here we have investigated pathway activities in dependence of different media compositions and mapped out differences in response to targeted therapies and distinct growth media. We assessed the response of PD3D® models derived from colorectal carcinoma (CRC) patients towards two targeted drugs aiming at different levels of the EGFR/Ras/Raf/Mek/Erk axis in defined, serum-free culture media. One media composition contained growth factors in concentration prevalently used in published cell culture protocols, while the other composition is geared to better match physiological levels of relevant supplements. We observed a significant impact of media conditions on sensitivity towards targeted cancer drugs. However, these differences were as individual as the models and the sensitivity against the treatment. To uncover the underlying mechanisms in the observed discrepancy we used DigiWest - a targeted proteomics technology - to assess pathways activity in the different models. We applied a panel of over 250 (phospho-)antibodies against proteins in the MAPK/ERK/RAS, PI3K/AKT and mTOR pathways as well as cell cycle and proliferation. Using functional pathway analysis, we mapped out the difference in response to targeted therapies and distinct growth media. In conclusion, media composition has great impact on the response to targeted drugs in vitro and therefore needs to be harmonized to ensure relevant results. Combination of PD3D® models and DigiWest functional pathway analysis represents a powerful tool to identify the molecular mechanisms underlying differential drug response. Further development of this collaborative approach will lead to a better understanding of the drug resistance or sensitivity and potentially even identification of signatures related to them. Citation Format: Jürgen Loskutov, Gerrit Erdmann, Anja Arndt, Quirin Graf Adelmann, Przemyslaw Dudys, Marie Flechner, Madeleine Nadolny, Katja Osman, Ulrike Pfohl, Christoph Reinhard, Markus Templin, Katja Uhlig, Lena Wedeken, Christian Regenbrecht. Let’s get physiological! Impact of media conditions on drug response to targeted therapies in CRC organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 187.