In recent years, the needs of mass spectrometry for biological samples have rapidly increased. In this study, in order to analyze water-containing biological samples using time-of-flight secondary ion mass spectrometry (TOF-SIMS), a rapid-cooling method, sectioning system, and a sample introduction method that could avoid frost formation in plant biological samples were developed. It was confirmed that both frost formation and evaporation of volatile compounds were prevented by a rapid-cooling and new introduction method. Essential elements and H2O could be detected by this new rapid-cooling TOF-SIMS methodology. Therefore, SIMS analysis in the natural state became possible.