BI-9321 is a potent, selective, and cellularly active NSD3-PWWP1 antagonist with a Kd of 166 nM. BI-9321 is not valid for NSD2-PWWP1 and NSD3-PWWP2. BI-9321 specifically disrupts the interaction of NSD3-PWWP1 with histones in U2OS cells with IC50 of 1.2 μM.