X-ray diffraction imaging (XDI) experiments at cryogenic temperatures allows us to analyze the internal structures of frozen-hydrated biological cells and cellular organelles. As biological specimens are easily degraded by drying, a humidity-controlled chamber is developed to maintain a moist atmosphere during specimen preparation. In the chamber, specimen particles are dispersed onto windows of thin silicon nitride membranes. Next, the particles are flash-cooled to a frozen-hydrated state using liquid ethane. This cryogenic method of specimen preparation allows us to harvest a large number of biological cells and isolated cellular organelles at desired periods in the cell cycle. Subsequently, the prepared specimens can be stored in liquid nitrogen until the use. In this chapter, the methods and devices developed for preparing frozen-hydrated biological specimens are described. In addition, the characteristics of frozen-hydrated specimens are described.