Ion suppression from the tissue matrix has a severe effect on the mass spectrometry imaging (MSI) of drugs. This problem hinders further applications of MSI in preclinical drug research and development. In this study, an in situ hydrogel conditioning method was developed to enhance the sensitivity of air-flow-assisted desorption electrospray ionization (AFADESI)-MSI. Instead of the traditional washing or digestion treatment in solvent, this method used a solid phase hydrogel to "wash" tissue sections. It was demonstrated that this in situ hydrogel conditioning method improved the drug signal by as much as 2- to 25-fold in MSI, especially for hydrophobic compounds. Furthermore, the obvious dislocation of analytes was not observed. The evaluation of spatial resolution indicated that the amount of dislocation in tissue sections with the hydrogel process was less than the resolution of AFADESI-MSI. The underlying reasons for the MSI signal enhancement were initially investigated. The decreased signal intensities of choline, betaine, and carnitine and the increased intensities of the [M + H]+/[M + Na]+ and [M + H]+/[M + K]+ ratios for drugs in the mass spectra of pretreated tissues provided evidence that this method can reduce the levels of highly competitive quaternary ammonium and inorganic salts in the tissues. The preformation of a thin liquid film for droplet pickup would also raise the ionization efficiency of drugs. These results demonstrated that this in situ hydrogel conditioning method provides a rapid and feasible approach to improving the sensitivity of ambient MSI for drug mapping in tissues.