<p>While an immense number of natural products have been screened for bioactivity using a variety of phenotypic assays, elucidation of their protein targets remains a significant bottleneck limiting further clinical research. Next to conventional pulldown assays and modern developments, such as cellular thermal shift assays (CETSA), yeast three-hybrid (Y3H) has emerged as a powerful yet underutilized tool that provides a low-barrier avenue for target identification of the challenging natural products space. Still, adoption of Y3H in natural products research has been slow in spite of successful target identification of a diverse range of small molecules reported in literature. In this perspective, we present the underlying methodology and its limitations as well as possible future developments and how it may accelerate and complement the target ID of natural compounds. We encourage natural product researchers to increase efforts on target elucidation of their lead compounds and propose Y3H as an approach, which could be integrated into existing workflows.</p> Graphical abstract <p></p>