Mixed-lineage kinase 3 (MLK3) plays a crucial role in the progression of several cancers and neurodegenerative disorders, positioning it as a promising target for drug discovery. However, current MLK3 inhibitors face limitations such as moderate potency, off-target activity against other kinases and suboptimal pharmacokinetic properties. To address these challenges, we employed a structure-based drug design strategy to develop a series of 3H-imidazo[4,5-b]pyridine derivatives. In the absence of an experimentally determined crystal structure, we generated a homology model of MLK3 and used molecular docking to predict the binding interactions of the designed compounds with its active site. Subsequently, we synthesized and experimentally evaluated these compounds as MLK3 inhibitors. A significant number of compounds exhibited substantial inhibition of MLK3 in enzymatic assays. Furthermore, several compounds demonstrated favorable physicochemical properties, suggesting promising pharmacokinetic profiles. These findings validate our structure-based approach and highlight the potential of these compounds as promising lead candidates for the development of novel MLK3-targeted therapeutics.