Glioblastoma (GBM) is the most aggressive primary brain tumor, with a median survival of approximately one year and limited therapeutic options. Chitinase 3-like 1 (CHI3L1) is increasingly recognized as a promising target in GBM due to its role in tumor progression and immune modulation. In this study, we employed an in-house structure-based screening strategy (SpaceDock) to explore a virtual chemical space of 377 billion compounds for potential CHI3L1 inhibitors. Using a reaction-aware ligand design approach, 60 top-scoring virtual hits were synthesized, and 45 were obtained with sufficient purity for experimental testing. Primary screening by microscale thermophoresis (MST) identified nine hits. Compounds showing dose-dependent binding were subsequently analyzed using surface plasmon resonance (SPR), leading to the identification of compound with a dissociation constant ( ) of 19.11 µM. Importantly, demonstrated robust, dose-dependent efficacy in a multicellular 3D GBM spheroid model, significantly reducing spheroid viability and downstream STAT3 signaling. These results highlight as a promising drug candidate for modulating the CHI3L1-STAT3 axis and underscore the potential of structure-guided, reactivity-aware virtual screening of ultralarge chemical spaces to target non-enzymatic, conformationally dynamic proteins in complex cancer models.