The production of protein crystals suitable for high-resolution structural analysis critically depends on the quality, homogeneity, and functional integrity of the protein sample throughout the crystallization workflow. This chapter provides a comprehensive overview of key biochemical and biophysical parameters that govern successful protein crystallization, including protein purity, conformational and compositional homogeneity, structural integrity, and biological activity. We discuss the application of different complementary techniques – including chromatographic methods, mass spectrometry, circular dichroism, surface plasmon resonance, and X-ray crystallography – to systematically assess and optimize these parameters from initial sample preparation to final crystal formation and subsequent X-ray data collection. By integrating orthogonal characterization approaches, common issues such as aggregation, degradation, conformational heterogeneity, and loss of function can be identified and mitigated prior to crystallization trials, thereby improving reproducibility and diffraction quality. Furthermore, the chapter highlights alternative X-ray data collection methodologies such as room-temperature, in situ, and automated data collection that further enhances crystallographic workflows by reducing crystal handling, improving experimental efficiency, and enabling the capture of biologically relevant conformational states.