Platelet-rich plasma (PRP) stimulates tissue healing and centrifugation is the only method for PRP preparation. The purpose of the present study was to develop a method to separate platelets without centrifugation. We used 16 polymers of different chemical characteristics and mixed each polymer with fresh whole blood containing anticoagulant citrate-dextrose. Then, we observed blood cell separation. Focusing on the effective polymers and comparing our method with the conventional centrifugation method, we examined platelet recovery rate and P-selectin expression, which represents platelet activation. Poly-L-glutamic acid (PGA) and poly-L-aspartic acid (PAA) separated platelets efficiently; however, these polymers activated platelets. On the contrary, poly (2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB), which has phosphorylcholine residues mimicking a cellular membrane molecule, separated platelets moderately and the PMB-separated platelets were nearly inactive compared to the separation with the ordinary centrifugation and PGA. Conclusively, the present experiments demonstrated that without centrifugation platelets can be separated from whole blood with some water-soluble polymers, such as PGA, PAA, and PMB, and that PMB has an advantage not to activate platelets.