Abstract: The utility of artificial microRNA (amiRNA) to induce specific gene silencing has been reported in many plant species, but silencing efficiency of differently designed amiRNA constructs in transgenic plants is less predictable. Thus, pre-validation of the silencing efficiency of designed amiRNA constructs is indispensable. In this study, to target the mRNA of SmPAP1, a R2R3-MYB transcription factor gene of Salvia miltiorrhiza, two amiRNAs were designed using WMD3 (Web MicroRNA Designer), designated as amiRNA1-SmPAP1 and amiRNA2-SmPAP1, respectively. The transient co-expressions of the two amiRNAs constructs combined with the 35S∶SmPAP1 plant over-expression vector were subsequently examined by Agrobacterium-mediated transformation into tobacco leaf cells, respectively. Results showed that the expression level of amiRNA2 was almost twice that of amiRNA1, and the silencing strength of SmPAP1 by amiRNA2 was 2.5 times higher than that by amiRNA1. The significant negative correlation between amiRNA abundance and expression level of SmPAP1 at both the mRNA and protein level was observed in the transient agro-infiltration assays. Therefore, the assay for the transient expression of amiRNA in tobacco leaf cells can rapidly and effectively pre-validate silencing efficiency of diverse designed amiRNAs, and provide an important reference for subsequent genetic transformation in plants.