The first microfluidic microphysiological systems (MPS) entered the academic scene more than 15 years ago and were considered an enabling technology to human in vitro (patho)biology and, therefore, to provide alternative approaches to laboratory animals in pharmaceutical drug development and academic research. Currently, the field generates more than a thousand scientific publications per year. Despite the MPS hype in academia and by platform providers, which say this technology is about to reshape the entire in vitro culture landscape in basic and applied research, MPS approaches neither have been widely adopted by the pharmaceutical industry yet nor have they reached regulated drug authorization processes. Here, 46 leading international experts from all stakeholder groups – academia, MPS supplier industry, pharmaceutical and consumer products industries, and leading regulatory agencies – analyzed challenges and hurdles along the MPSbased assay life cycle in the second workshop of its kind in June 2019. The main findings were that the level of qualification of MPS-based assays for a given context of use and communication gaps between stakeholders are the major challenges slowing industrial adoption by end users, which in turn is causing a regulatory acceptance dilemma. This report elaborates on these findings and proposes solutions by providing recommendations and a roadmap towards regulatory acceptance of MPS-based models, which will benefit patients and further reduce laboratory animal use in drug development. Finally, the potential of MPS-based human disease models to feed back into laboratory animal replacement in basic life science research is discussed.