<p>Hepatocellular carcinoma (HCC) is one of the most frequent causes of cancer-related death worldwide. Mirroring the complexity of human HCC with its underlying liver disease requires multilocular orthotopic tumor models in mice, where objective tumor quantification in vivo is challenging, especially longitudinally. We investigated magnetic resonance imaging (MRI) to noninvasively quantify orthotopic HCC in an immunotherapy setting. Orthotopic HCCs were induced in diethylnitrosamine (DEN)-injected mice with either CCl₄-induced hepatic fibrosis or dietary metabolic dysfunction-associated steatotic liver disease (MASLD) to mirror the most frequent etiologies. Growth kinetics over the course of immune checkpoint inhibitor treatment with anti-Programmed Death Ligand 1 (αPD-L1) were modeled by measuring the overall tumor burden (OTB) using MRI and compared to untreated animals. Tumor parameters, such as tumor volume, liver weights at sacrifice and largest tumor diameter were analyzed. We demonstrate that MRI is a reliable imaging tool for both pretreatment tumor confirmation as well as the longitudinal quantification of overall tumor burden under investigational treatment. While measuring only the largest tumor diameter yielded significant differences between αPD-L1-treated animals and untreated controls in the long-term setting, these trends in tumor response were not confirmed by MRI-based OTB measurement. In MASLD-HCC, tumors did not respond to PD-L1 blockade, thus confirming the OTB data and reflecting the immunotherapy resistance observed in the human setting and highlighting the translational relevance of the model. Orthotopic fibrosis-HCC and MASLD-HCC mouse models can be enhanced through the longitudinal use of MRI while reducing endpoints and animal numbers. The DEN-CCl₄ and DEN-Western Diet models mirror the αPD-L1 response from the human setting and require OTB measurement, because largest diameters may underestimate the total tumor mass in orthotopic HCC.</p>