The p21-activated kinases (PAKs) are a family of serine/threonine protein kinases and activated by binding with activated Rho GTPases such as Rac or Cdc42. PAKs regulate actin cytoskeletal remodeling, cell motility, cell survival, and apoptosis. Also, PAKs are involved in several diseases such as cancer, virus infectious diseases, mental retardation, Alzheimer and Parkinson's diseases. Therefore, the substances that are able to inhibit PAK activation can be used as powerful tools and medicines for PAK relative diseases or specific inhibitors for study of PAK signaling pathway. In this study, we investigated and characterized the 5 compounds of 4-benzene-1, 2-naphthoquinone (NQ) family as candidate substances to inhibit the PAK1 activation in vitro and in cells. Binding between p21-binding domain (PBD) of PAK1 and Cdc42 was blocked by 5 NQ-compounds in ELISA assay. Myelin basic protein (MBP) phosphorylation was dramatically reduced by treatment of these compounds in vitro kinase assay for Cdc42-induced or constitutive active PAK1 mutant. Also, phosphorylation at Thr 423 of transfected PAK1 was inhibited by treatment of 5 NQ-compounds in 293T cells, respectively. Finally, NQ-5 inhibited strongly the PAK1 activation by PDGF stimulation and cell motility in PDGF-induced wound migration assay in NIH 3T3 cells. Therefore, these NQ compounds will be good candidates as target molecules to regulate PAK1-related diseases or inhibitors to study PAK1 signaling pathway.