Raman spectroscopy is a powerful analytical technique with significant potential in preclinical bioimaging due to its non-invasive and label-free molecular insights. This chapter explores its diverse applications in advancing biomedical research and drug development, highlighting unique advantages over traditional imaging methods, such as real-time monitoring, chemical specificity, and compatibility with living tissues. Technological advancements, including confocal Raman microscopy, Raman imaging, Surface-Enhanced Raman Spectroscopy (SERS), and Tip-Enhanced Raman Scattering (TERS), have expanded its applications in mapping biochemical processes and identifying disease-specific biomarkers. The integration of Raman spectroscopy with complementary imaging modalities, such as fluorescence microscopy, atomic force microscopy, and mass spectrometry, underscores its potential to provide comprehensive insights into cellular and tissue-level dynamics. Case studies demonstrate its versatility, with applications ranging from cancer research to neurodegenerative diseases and nanoparticle-based drug delivery, effectively monitoring tumor progression, drug biodistribution, and molecular changes associated with diseases. Furthermore, Raman spectroscopy is progressing toward clinical applications, driven by technological advancements addressing weak signal intensity and in vivo complexity. Notable developments include endoscopic Raman systems, which combine high chemical specificity with minimally invasive tools for real-time tissue assessment in oncology and other fields. In conclusion, Raman spectroscopy holds great promise for transforming preclinical bioimaging, offering detailed molecular insights that complement existing imaging modalities. Continued technological innovation is expected to bridge the gap between basic research and clinical applications, accelerating the translation of biomedical discoveries into therapeutic innovations.