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Quantitative preclinical imaging enables non-invasive characterization of physiological, molecular, and functional processes across a variety of experimental models, providing metrics that inform longitudinal studies and translational research. This review synthesizes current strategies for quantitative imaging across modalities including Positron emission tomography (PET), Single Photon Emission Computed Tomography (SPECT), Magnetic resonance imaging (MRI), Computed Tomography (CT), optical imaging, and hybrid systems. We examine methodological frameworks for parameter extraction, reproducibility, and validation against biological reference standards, evaluating each modality through a cross-cutting analytical framework that distinguishes technical, biological, and computational sources of quantitative variance and identifies the current metrological maturity of harmonization infrastructure across platforms. Key challenges, such as protocol harmonization, cross-platform comparability, and integration across species, are analyzed, alongside computational advances including parametric mapping, and artificial intelligence–assisted pipelines. Emerging approaches that combine multimodal acquisition with standardized reconstruction and calibration strategies are also discussed, emphasizing their potential to enhance precision, reduce bias, and support biologically meaningful interpretation. Collectively, this review provides a comprehensive perspective on the design, implementation, and validation of quantitative preclinical imaging studies, offering practical guidance for generating reproducible, interpretable, and translationally relevant imaging biomarkers. By integrating methodological insights with advances in technology and analytics, it underscores the role of quantitative frameworks in bridging preclinical discovery with translational applications.