Abstract: The objective of the study was to investigate the effects of nebulizers and add-on spacers on nebulization performances of Re-Du-Ning injection. Chlorogenic acid and geniposide were taken as the chemical markers of the injection. In vitro nebulization performances including real-time particle size distribution, aerodynamic particle size distribution (APSD), respirable drug delivery rate (RDDR) and percentage of respirable delivered dose (RDD%), residual rate and nebulization time were determined after nebulization of Re-Du-Ning injection via jet or mesh nebulizers. The delivery efficiency was the ratio of delivery dose to the drug content of the injection. The aerosol delivery efficiency of Re-Du-Ning injection for the jet nebulizer was about 30% of the nominal dose whereas those for mesh nebulizers varied from 24% to 35% in adult breathing pattern. Upon the inclusion of spacers, the delivery efficiencies of mesh nebulizers increased to 37%-68% whereas the presence of a spacer decreased the delivery efficiency of the jet nebulizer. When the aerosols of mesh nebulizer exhibited comparative fine particle fraction (FPF) and mass median aerodynamic diameter (MMAD) to those of the jet nebulizer, the RDD% of the former nebulizer with a spacer was more than two folds of the latter. These results indicated that the addition of spacer was demonstrated to significantly improve the delivery efficiency of Re-Du-Ning injection by mesh nebulizers.