Single-point mutations of certain residues (so-called hot spots) impair/disrupt protein-protein interactions (PPIs), leading to pathogenesis and drug resistance. Conventionally, a PPI-hot spot is identified when its replacement decreased the binding free energy significantly, generally by ≥2 kcal/mol. The relatively few mutations with such a significant binding free energy drop limited the number of distinct PPI-hot spots. By defining PPI-hot spots based on mutations that have been manually curated in UniProtKB to significantly impair/disrupt PPIs in addition to binding free energy changes, we have greatly expanded the number of distinct PPI-hot spots by an order of magnitude. These experimentally determined PPI-hot spots along with available structures have been collected in a database called PPI-Hotspot. We have applied the PPI-Hotspot to create a nonredundant benchmark, PPI-Hotspot+PDB, for assessing methods to predict PPI-hot spots using the free structure as input. PPI-Hotspot will benefit the design of mutagenesis experiments and development of PPI-hot spot prediction methods. The database and benchmark are freely available at https://ppihotspot.limlab.dnsalias.org.