The recent publication of a potent and selective inhibitor of protein methyltransferase 5 (PRMT5) provides the scientific community with in vivo-active tool compound <b>EPZ015666b> (<b>GSK3235025b>) to probe the underlying pharmacology of this key enzyme. Herein, we report the design and optimization strategies employed on an initial hit compound with poor in vitro clearance to yield in vivo tool compound <b>EPZ015666b> and an additional potent in vitro tool molecule <b>EPZ015866b> (<b>GSK3203591b>).