Rho kinase (ROCK) inhibitors are effective candidates for neural or cardiovascular disorders. High throughput model for screening ROCK inhibitors is a basic foundation to pick up ROCK inhibitors from thousands of compounds for drug developing. The high throughput model was established based on purified recombinant rat ROCK catalytic domain (rROCK-CD) from Escherichia coli (E. coli). There are two steps of reaction in the model: incubation of 5.0 microl recombinant rROCK-CD (2.0 microg/ml), 5.0 microl different compounds, 5.0 microl fluorescent S6-peptide (200 nM), and 5.0 microl ATP (10 microM) at 37 degrees C for 60 min was made the first reaction, and the second reaction was made by incubating them with additional 60 microl binding reagent at ambient temperature for 30 min. The phosphorylated S6 peptide can bind to a binding reagent, and the fluorescence varies from low polarization to high according to the amount of the phosphorylated peptide. IC50 was calculated based on polarization variation. Compound, which IC50 was less than 10 microM, was recognized as a lead compound which taken bioactivity evaluation in PC12 by observing neurite outgrowth. The Z'-factor of the model is 0.81 (above 0.5). The model screened five lead compounds from 3294, which promoted neurite outgrowth to different extent. The results suggested that the model is suitable for high throughput screening (HTS), and the five lead compounds are worth of further investigation.