Photoacoustic Imaging (PAI) is a cutting-edge imaging modality that combines the advantages of optical and ultrasound imaging, offering high-contrast and deeper penetration in order to visualize and extract anatomical, functional, and molecular information of the biological tissues. Due to the noninvasive nature and easy integration with popular ultrasound diagnostic imaging, PAI has been utilized for several applications in preclinical studies, such as vascular imaging, utilizing endogenous contrast agents’ characteristics like hemoglobin to measure parameters such as blood oxygenation and flow velocity. Major configurations of PAI i.e., photoacoustic tomography and photoacoustic microscopy excel in imaging small vessels and microvasculature, enabling the study of angiogenesis, tumor malignancy, ischemic conditions, etc. PAI’s ability to differentiate oxygenated and deoxygenated hemoglobin provides critical insights into tissue states, particularly for stroke and tumor imaging. Advanced systems such as multi-spectral optoacoustic tomography and spiral volumetric optoacoustic tomography enable 3D imaging with enhanced resolution and speed. PAI also integrates exogenous contrast agents, including nanoparticles and dyes, to enhance imaging specificity for targeted molecular and functional studies. Its applications range from tumor imaging, wound healing monitoring to drug delivery assessment. By merging functional and molecular imaging capabilities, PAI demonstrates its versatility in preclinical research and its potential for clinical application.