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Serial femtosecond crystallography (SFX) enables the determination of the room‐temperature crystal structure of macromolecules, as well as providing time‐resolved molecular dynamics data in pump–probe experiments. Fixed‐target SFX (FT‐SFX) can minimize sample consumption and physical effects on crystals during sample delivery. In FT‐SFX studies, having a sample holder that can stably fix crystal samples is one of the key elements required for efficient data collection. Hence, development of sample holders from new materials capable of supporting various crystal sizes and shapes may expand the applications of FT‐SFX. Here, a viscous‐media‐based crystal support in a sample holder for FT‐SFX is introduced. Crystal samples were embedded in viscous media, namely gelatin and agarose, which were enclosed in a polyimide film. In the vertically placed sample holder, 10–15%(w/v) viscous gelatin and 1–4%(w/v) agarose gel stably supported crystals between two polyimide films, thereby preventing the crystals from descending owing to gravity. Using this method, FT‐SFX experiments were performed with glucose isomerase and lysozyme embedded in gelatin and agarose, respectively. The room‐temperature crystal structures of glucose isomerase and lysozyme were successfully determined at 1.75 and 1.80 Å resolutions, respectively. The glucose isomerase and lysozyme diffraction analyses were not impeded by excessive background scattering from the viscous media. This method is useful for delivering crystal samples of various sizes and shapes in FT‐SFX experiments. [ABSTRACT FROM AUTHOR]