The hazardous effects due to the insoluble microparticles generated in the injections have been pointed out. To our knowledge, however, there have been no reports about insoluble microparticulate contamination at ampoule opening. Therefore, we performed this study to evaluate the relationship between time and glass particulate sedimentation to examine the effect of swabbing the ampoule neck on particle generation to clarify the relationship between the inner-diameter size of an ampoule and the amount of glass particulate sedimentation to find out the effect of methods for ampule opening, and compare particle contamination in glass ampoule and that of plastic, after ampoule opening and assessed the contribution of material of ampoule to the particle generation. We observed that the glass particulate contamination of accumulation value at a size over 2 μm increased significantly after 60 seconds, the swabbing the neck of the ampoule prior to opening had a negative effect on prevention of glass particulate contamination, the glass particulate contamination was positively influenced by the inner-diameter size of the ampoule, but not by the thickness of the ampoule walls, the particulate contamination derived from glass significantly increased by general method as well as using ampoule open adaptor compared with our method, and the insoluble microparticulate contamination in plastic ampoule was significantly lower than that in glass ampoule. The present findings might provide an useful information to reduce glass particules after ampoule opening performed in clinical practice.