Direct tissue analysis and imaging using mass spectrometry can extract a mass of information directly from complex biological samples. To carry out successful profiling and mapping of biomolecules, though, techniques for correct sample preparation are required. This article focuses on the influences of tissue thickness on mass spectra. We obtained the mass spectra and imaging results as a function of thickness from 2 to 40 μm, then evaluated the spectrum quality. The peak intensity and number of observed peaks drastically increased as the sections became thinner (<10 μm). Significant images of high molecular weight proteins were acquired only from the thinner slices. We thus concluded that the thickness of biological tissue slices was an important factor in obtaining high quality mass spectra. There could be two reasons for inefficient analyte ionization of thicker slices: electrical nonconductive properties and impurities in thick tissue sections. In conclusion, this investigation extends the capability of imaging mass spectrometry, especially for high molecular weight protein imaging, by optimizing the thickness of the tissue samples.