Key parameters of air flow assisted ionization mass spectrometry imaging（AFAI-MSI） system were investigated and optimized by using tissue sections adding homogeneous NHBA as the target samples.The parameters，including those related with spray solvent，spray gas，as well as geometric setting of AFAI system were optimized. Based on the optimization，stability of endogenous metabolites from the tissue section was also taken into consideration during the AFAI-MSI experiment.It usually takes 7-8 h for a complete MSI experiment for a whole-body tissue section，and the stability of endogenous metabolites needs to be investigated during such a long process. Therefore，a whole-body tissue section was imaged twice consecutively，and then the data of resulted images were compared using visualized component principle analysis（PCA） after selecting main mass spectra peaks.By visualized comparing of the result，the similarity of the visualized PCA score plot demonstrated the endogenous metabolites were stable during MSI experiment.In conclusion，the optimized parameters guaranteed the stability and sensitivity of AFAI-MSI experiment.Additionally，after proper sample pretreatment，the endogenous metabolites in tissue sections were able to remain stable during the AFAI-MSI experiment，which ensured the reliability for research of the variability of endogenous metabolites.Thus the AFAI-MSI method is promising to be applied in the study of drug development.