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Understanding the enzyme reaction mechanism is important for identifying fundamental biochemical processes and for providing invaluable information for enzyme engineering for industrial applications. Serial femtosecond crystallography (SFX) enables determination structure of enzymes without radiation damage at room temperature. Time-resolved SFX with ligand mixing provides a molecular movie of enzyme reactions, including the transition states. This structural information provides insight into previously undiscovered molecular dynamics over various time periods. To observe enzyme reactions with SFX, it is essential to comprehend the device used for mixing the protein crystals and reaction solutions, as well as the mixing efficiency and available time delay provided by this equipment. In this review, we summarize the current mixing techniques for time-resolved SFX. We describe the configuration and experimental setup of the SFX sample environment unique to each experimental technique and discuss the technical possibilities and limitations of these techniques for performing time-resolved crystallography. • Time-resolved mixing SFX experiments for enzyme reactions are explained. • Mixing devices and time-resolved mixing SFX results are summarized. • Potential available mixing devices for time-resolved SFX are discussed. • The possibilities and limitations of time-resolved mixing SFX are discussed. [ABSTRACT FROM AUTHOR]