X-ray crystallography determines the atomic and molecular structures of crystalline solids by analyzing the diffraction patterns of X-rays interacting with crystal lattices. This method gives three-dimensional electron density maps that reveal atomic positions, chemical bonds, and crystallographic features, making it indispensable across chemistry, biology, materials science, and pharmaceuticals. Since its development in the early 20th century, X-ray crystallography has enabled critical discoveries such as the determination of DNA’s double-helix structure and the characterization of numerous proteins, enzymes, and drug molecules. Its ability to provide high-resolution structural insights continues to support fields ranging from mineralogy to drug discovery, guiding innovations in rational drug design and materials engineering. Advances in instrumentation and computational methods have expanded the scope and precision of crystallography. The development of synchrotron radiation, X-ray free-electron lasers (XFELs), and serial femtosecond crystallography (SFX) has enabled the study of micro- and nanocrystals, reduced radiation damage, and allowed time-resolved imaging of dynamic processes at atomic resolution. These innovations have not only enhanced traditional small-molecule and macromolecular crystallography but also opened new avenues such as quantum crystallography and integrative approaches combining X-ray data with cryo-electron microscopy and NMR. Despite challenges such as the need for high-quality crystals and large datasets, X-ray crystallography remains the gold standard for structural determination, providing unmatched insights into the architecture and function of matter.