Optical imaging is crucial in preclinical evaluation of tumor development, progression, and therapies, due to its low cost, versatility, sensitivity, and capability of real-time monitoring. The main in vivo imaging techniques include fluorescence, bioluminescence, and photoacoustic imaging. Although all three applications are limited by relatively low tissue penetration due to absorption and scattering of light in biological tissues, each application has its own benefits. Fluorescence imaging is technically the simplest method to perform and using near infrared fluorescent (NIRF) probes can enhance depth capability. Moreover, novel activatable probes, lifetime imaging, and multimodal imaging strategies have extended its utility to endoscopic and surgical procedures. Bioluminescence imaging boasts high sensitivity and signal-to-noise ratio, as light emission originates at the target from enzymatic reactions. However, it requires genetic modification to express luciferases, restricting its use in humans. Photoacoustic imaging offers better depth penetration compared to purely optical methods by utilizing ultrasound signals. Leveraging endogenous contrast from chromophores, it assists in vascular imaging and tumor identification without external labels. Due to the use of safe, non-ionizing radiation, it reduces the risk to subjects and allows repeated imaging, and is thus most suited for clinical translation. All three applications continuously benefit from the development of three-dimensional imaging techniques and deep learning algorithms.