Time-resolved serial femtosecond crystallography (tr-SFX) enables the study of biomolecules in action at room temperature, thus facilitating the construction of in crystallo mini-3D biomolecular series (a stop-motion series) of biochemical reactions with unprecedented spatiotemporal details. One of the challenges encountered in expanding this method is the insufficient tools available for triggering the biomacromolecular reaction. Here, we highlight recent advances and challenges in the mix-and-inject (diffusion-based) tr-SFX (MISC) as a promising triggering method for studying the structural dynamics of metalloproteins, redox enzymes, and their reaction kinetics. We further discuss the results obtained using MISC tr-SFX and propose complex MISC (cMISC) as a tool to study complex reaction kinetics such as theenzyme-catalyzed bisubstrate (sequential and ping-pong) reactions.