OBJECTIVE： To optimize the best crushing particle size of Agkistrodon decocting powder， and study its effect on apoptosis of human rheumatoid arthritis fibroblast-like synoviocytes. METHODS： Pre-column derivatization RP-HPLC was adopted. Using the total decoction amounts of 4 main amino acids （aspartic acid， glutamic acid， L-hydroxyproline， glycine） that decocted once as index， the Agkistrodon decoction pieces and those through No.1-8 seive were screened， and the best crushing particle size of Agkistrodon decocting powder was optimized. The human rheumatoid arthritis fibroblast-like synoviocytes were divided into negative control group， positive control group （1 μmol/L methotrexate） and the best crushing particle size of Agkistrodon decocting powder group （2.0 mg/mL）， flow cytometry was used to determine the cell apoptosis after cultured for 48 h. RESULTS： The best crushing particle size was through No.6 seive， when the total amount of 4 main amino acids was （61.27±0.02） mg/g （n＝3）. Compared with negative control group， the apoptosis rate in the best crushing particle size of Agkistrodon decocting powder group was significantly increased （P＜0.05）， which was slightly higher than positive control group. CONCLUSIONS： The best crushing particle size is through No.6 seive； and Agkistrodon decocting powder shows effect on reducing the apoptosis of human rheumatoid arthritis fibroblast-like synoviocytes.