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Functional characterization of proteins requires insights into the interplay between structure and dynamics. Classical approaches to X-ray crystallography via populating intermediate states by mutation or chemical trapping only provide static time-averaged data sets. Acquiring dynamic data is thus highly important. However, elucidating models of protein dynamics that are related to functional characteristics is often rife with difficulty, as enzyme systems are highly complex, and require structural data as a function of time where key insights can be obtained by visualizing the wide range of both internal motions and interactions with the solvent environment. Considering this, serial time-resolved approaches are powerful tools to elucidate time-dependent structures. To this end we have combined and established novel fixed target approaches which enable unique data collection strategies, such as the “Hit And REturn” (HARE) approach. As well as novel reaction initiation strategies with the use of piezo droplet injectors. The “Liquid Application Method for time-resolved Analysis” (LAMA) method simplifying reaction initiation for systems that are not naturally amenable to light activation. Building on these advancements, we have also developed the spitrobot, a protein crystal plunge-freezer. This device enables cryo-trapping with millisecond resolution, allowing for the observation of ligand binding, reaction intermediates, and conformational changes during enzymatic turnover. Furthermore, we have developed an environmental setup that allows for the modification of temperature during these observations. We demonstrate binding of several ligands in two enzymes, and trapping of several reaction intermediates and conformational changes during the turnover, as demonstrated with the enzyme Xylose isomerase, where we can monitor the glucose to fructose conversion over time and across a range of temperatures. Further advances in sample handling and data acquisition strategies are enabling us to delve further into the regulatory mechanisms of allostery and water networks for proteins, inevitably providing insights into fundamental enzymology.