Structural biology has played a pivotal role in the major scientific discoveries, enhanced our understanding of diverse molecular mechanisms, and led to the discovery of new therapeutics. Here, we discuss the various approaches employed to determine the structure of biological macromolecules, their limitations, and recent advancements in the field. X-ray crystallography remains the most preferred method for the structural characterization of biological macromolecules, despite the limitations of obtaining diffraction-quality crystals and beam-induced radiation damage. Recent advances in methodologies like X-ray free electron lasers (XFEL) and small-angle X-ray scattering (SAXS) assist in high-intensity diffraction studies and solution-based structural analysis, respectively. A key technique for studying macromolecular structures at atomic resolution is nuclear magnetic resonance (NMR) spectroscopy. NMR is critical for studying protein dynamics, ligand interactions, and membrane–protein structures. Another crucial advancement in the field, cryo-electron microscopy or cryo-EM, helps preserve macromolecular structures in a “near-native” state by rapid freezing and has been tremendously successful in obtaining structures of large macromolecular complexes. Cryo-electron tomography (cryo-ET) and micro-electron diffraction (micro-ED) have extended the capabilities of cryo-EM in providing a detailed cellular context and elucidating structural details from tiny crystalline samples (<500 nm). In combination, all these techniques work wonderfully to obtain more accurate structural models. The improvements in data-processing software for single-particle analysis (SPA) and the emergence of direct electron detectors have been instrumental in bringing the resolution revolution in cryo-EM. The integration of machine learning and artificial intelligence (AI)-based tools such as AlphaFold, RoseTTAFold, OmegaFold, and ESMFold has transformed the field of computational structural biology. These tools help predict the macromolecular structures more accurately. The chapter delves into these recent advancements while discussing the future course of macromolecular structure determination.