<p>Three-dimensional protein structure determination by X-ray crystallography is essential for understanding biological function and accelerating drug discovery. However, obtaining high-quality protein crystals remains a significant bottleneck. The conventional crystallization methods are often labor-intensive, require large sample volumes, and offer limited control over the crystallization environment. This review summarizes the application of microfluidic technologies to protein crystallography with a focus on their advantages over the conventional crystallization methods. Microfluidic devices enable nanoliter-scale sample handling, precise control over crystallization conditions, and high-throughput screening, addressing major limitations of the conventional approaches. This review introduces various microfluidic platforms, including droplet-based and microwell-based systems, for protein crystallization, crystal growth control, and on-chip X-ray diffraction analysis. The review also covers the use of microfluidics for creating diffusion-controlled crystal growth environments, real-time crystal growth measurement, on-chip X-ray diffraction measurement, and room-temperature X-ray crystallography with automated data processing.</p> Graphical abstract <p></p>