Protein crystallization is the process of formation of a regular array of individual protein molecules stabilized by crystal contacts. If the crystal is sufficiently ordered, it will diffract. Some proteins naturally form crystalline arrays, like aquaporin in the lens of the eye.

In the process of protein crystallization, proteins are dissolved in an aqueous environment and sample solution until they reach the supersaturated state. Different methods are used to reach that state such as vapor diffusion, microbatch, microdialysis, and free-interface diffusion. Developing protein crystals is a difficult process influenced by many factors, including pH, temperature, ionic strength in the crystallization solution, and even gravity. Once formed, these crystals can be used in structural biology to study the molecular structure of the protein, particularly for various industrial or medical purposes.

For over 150 years, scientists from all around the world have known about the crystallization of protein molecules.

In 1840, Friedrich Ludwig Hünefeld accidentally discovered the formation of crystalline material in samples of earthworm blood held under two glass slides and occasionally observed small plate-like crystals in desiccated swine or human blood samples. These crystals were named as 'haemoglobin', by Felix Hoppe-Seyler in 1864. The seminal findings of Hünefeld inspired many scientists in the future.

In 1851, Otto Funke described the process of producing human haemoglobin crystals by diluting red blood cells with solvents, such as pure water, alcohol or ether, followed by slow evaporation of the solvent from the protein solution. In 1871, William T. Preyer, Professor at University of Jena, published a book entitled Die Blutkrystalle (The Crystals of Blood), reviewing the features of haemoglobin crystals from around 50 species of mammals, birds, reptiles and fishes. These early approaches relied on simple evaporation techniques and worked mainly with naturally abundant proteins such as hemoglobin.

In 1909, the physiologist Edward T. Reichert, together with the mineralogist Amos P. Brown, published a treatise on the preparation, physiology and geometrical characterization of hemeoglobin crystals from several hundreds animals, including extinct species such as the Tasmanian wolf. Increasing protein crystals were found. Between 1909 and the 1930s, scientists crystallized enzymes (urease by Sumner, 1926; pepsin by Northrop, 1929; and trypsin/chymotrypsin later). These crystallizations were crucial because they proved enzymes are proteins, overturning a major debate. Around the same period, the development of “salting out” with ammonium sulfate allowed scientists to deliberately crystallize enzymes. In 1926, James B. Sumner crystallized urease, proving for the first time that enzymes are proteins, and this was soon followed by John H. Northrop’s crystallization of pepsin in 1929.

In 1934, John Desmond Bernal and his student Dorothy Hodgkin discovered that protein crystals surrounded by their mother liquor (the remaining solution after a protein has crystallized out of a supersaturated solution) gave better diffraction patterns than dried crystals. Using pepsin, they were the first to discern the diffraction pattern of a wet, globular protein. Prior to Bernal and Hodgkin, protein crystallography had only been performed in dry conditions with inconsistent and unreliable results. This is the first X‐ray diffraction pattern of a protein crystal.

Bernal and Hodgkin's findings marked the beginning of modern protein crystallography, demonstrating that proteins could yield interpretable diffraction patterns suitable for structure determination. This success encouraged further attempts at applying X-ray diffraction to biological macromolecules. In the late 1930s, Bernal’s group and others refined methods for mounting and preserving crystals, while William Astbury and colleagues extended fiber diffraction studies to proteins such as keratin and myosin, foreshadowing later breakthroughs in structural biology.