This work describes the characterisation of innovative and reliable preclinical models of the malignant pleural mesothelioma. We developed one in vitro model of spheroids, one in ovo model of tumour xenograft on the chicken chorioallantoic membrane, and one in vivo model of orthotopic tumour xenograft in immunodeficient mice. We demonstrated a higher relevance of these models than the currently used in vitro monolayer cell culture and in vivo subcutaneous xenograft. We also monitored in vivo tumour growth and metabolic activity by [18F]FDG-microPET/CT non-invasive imaging. Overall, these innovative highly reliable models could be used in an efficient drug flow discovery linked to clinically relevant diagnostic strategies. These models demonstrate a high 3R potential (Reduce, Refine, Replace) and the described non-invasive molecular imaging techniques greatly promote and accelerate the translation of preclinical animal results to clinical settings.