The absent, small, or homeotic-like 1 (ASH1L) protein is a histone lysine methyltransferase that plays a crucial role in various cancers, including leukemia. Despite representing an attractive therapeutic target, only one class of ASH1L inhibitors was identified to date. Herein, we report development of advanced ASH1L inhibitors targeting the catalytic SET domain, which were designed to access previously unexplored binding pocket on ASH1L. Extensive medicinal chemistry combined with structure-based design led to identification of 66s (AS-254s), a highly potent and selective ASH1L inhibitor (IC50 = 94 nM), representing substantially improved inhibitory activity over previously reported compounds targeting ASH1L. Furthermore, 66s effectively blocked cell proliferation and induced apoptosis and differentiation in leukemia cells harboring MLL1 translocations. Overall, this work provides a high-quality chemical probe targeting the catalytic SET domain of ASH1L with increased inhibitory activity and cellular efficacy to study biological functions of ASH1L and potentially to develop novel anticancer therapeutics.