Here, we report the fragment-based drug discovery of potent and selective fragments that disrupt the Spire2-FMN2 but not the Spire1-FMN2 interaction. Hit fragments were identified in a differential scanning fluorimetry-based screen of an in-house library of 755 compounds and subsequently validated in multiple orthogonal biophysical assays, including fluorescence polarization, microscale thermophoresis, and 1H-15N HSQC nuclear magnetic resonance. Extensive structure-activity relationships combined with molecular docking followed by chemical optimization led to the discovery of compound 13, which exhibits micromolar potency and high ligand efficiency (LE = 0.38). Therefore, this fragment represents a validated starting point for the future development of selective chemical probes targeting the Spire2-FMN2 interaction.