The present paper aims at identifying strategies to increase the impact and applicability of alternative testing strategies in risk assessment. To this end, a quantitative and qualitative literature evaluation was performed on (a) current research efforts in the development of in vitro methods aiming for alternatives to animal testing, (b) the possibilities and limitations of in vitro methods for regulatory purposes and (c) the potential of physiologically-based kinetic (PBK) modeling to improve the impact and applicability of in vitro methods in risk assessment practice. Overall, the evaluation showed that the focus of state-of-the-art research activities does not seem to be optimally directed at developing in vitro alternatives for those endpoints that are most animal-demanding, such as reproductive and developmental toxicity, and carcinogenicity. A key limitation in the application of in vitro alternatives to such systemic endpoints is that in vitro methods do not provide so-called points of departure, necessary for regulators to set safe exposure limits. PBK-modeling could contribute to overcoming this limitation by providing a method that allows extrapolation of in vitro concentration–response curves to in vivo dose–response curves. However, more proofs of principle are required.