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Abstract Medical devices and technologies must undergo extensive testing and validation before being certified for public healthcare use, especially in oncology where a high research focus is on new advancements. Human 3D-tissue models can offer valuable insights into cancer behavior and treatment efficacy. This study developed a cell phantom setup using a rattail collagen-based hydrogel to facilitate reproducible investigations into ablation techniques, focusing on electroporation (EP) for lung tumor cells. The temperature rise due to the treatment is a critical aspect based on other studies that have discovered non-neglectable temperature values. A realistic physiological, biological phantom is crucial for electrode material development, non-thermal ablation control, tumor cell behavior study, and image-guided treatment simulation. The test system comprises a standardized 3D-printed setup, a cell-mimicking hydrogel model cultivated with NIH3T3 and HCC-827 cell lines. The treatment is evaluated with an AlamarBlue assay and the temperature is monitored with a sensor and a non-invasive MR-thermometry. Results showed the reliability of the selected monitoring methods and especially the temperature monitoring displayed interesting insights. The thermal effect due to EP cannot be neglected and it has to be discussed if this technique is non-thermal. The lesions in the phantom were able to show apoptotic and necrotic regions. The EP further led to a change in viability. These results suggest that the phantom can mimic the response of soft tissue and is a useful tool for studying cellular response and damage caused by EP or other treatment techniques.