EphB3 is a major key player in a variety of cellular activities, including cell migration, proliferation, and apoptosis. However, the exact role of EphB3 in cancer remains ambiguous. Accordingly, new EphB3 inhibitors can increase the understanding of the exact roles of the receptor and may act as promising therapeutic candidates. Herein, a hybrid approach of structure-based design and virtual combinatorial library generated 34 quinazoline sulfonamides as potential selective EphB3 inhibitors. A molecular docking study over EphB3 predicted the binding affinities of the generated library, and the top seven hit compounds (3a and 4a–f), with GlideScore ≥ −6.20 Kcal/mol, were chosen for further MM-GBSA calculations. Out of the seven top hits, compound 4c showed the highest MM-GBSA binding free energy (−74.13 Kcal/mol). To validate these predicted results, compounds 3a and 4a–f were synthesized and characterized using NMR, HRMS, and HPLC. The biological evaluation revealed compound 4c as a potent EphB3 inhibitory lead (IC50 = 1.04 µM). The screening of 4c over a mini-panel of kinases consisting of EGFR, Aurora A, Aurora B, CDK2/cyclin A, EphB1, EphB2, EphB4, ERBB2/HER2, and KDR/VEGFR2, showed a promising selective profile against EphB3 isoform. A dose-dependent assay of compound 4c and a molecular docking study over the different forms of EphB provided insights into the elicited biological activities and highlighted reasonable explanations of the selectivity.