Molecular imaging allows noninvasive visualization of biological processes in their native environment within living systems. It can give a better understanding of fundamental biology, help in identifying disease mechanisms and visualizing pathological tissues, and enable monitoring disease progression and studying the drug efficacy in intact living organisms. Among various imaging modalities, bioluminescence and fluorescence imaging are powerful techniques that have become essential methods for non-invasive real-time studies of biological processes in vivo. The aim of my thesis was the development of novel fluorescent and bioluminescent probes for in vivo imaging of fatty acid and triglyceride uptake. The goal of my first project was the development of a near infrared fluorescent fatty acid probe for glioma imaging. Some tumors such as glioma may rely on the uptake of extracellular fatty acids. Fatty acids can therefore be considered as targeting molecules for these tumors and could be used to develop tumor-imaging probes. The probe design is based on a near-infrared fluorophore indocyanine green (ICG) that is conjugated to a long-chain fatty acid palmitic acid. The conjugation of ICG to a fatty acid (ICG-FA) was envisioned to improve cell permeability of the fluorophore and its accumulation in glioma. The ICG-FA probe was first evaluated for its ability to mimic the uptake of natural fatty acids in cells and was then applied for glioma imaging studies in vivo, where it showed significant tumor accumulation. As a proof-of-concept, the probe was tested for intraoperative image-guided surgery in a canine patent with mastocytoma, where it allowed intraoperative fluorescence tumor imaging and provided optical guidance for a surgeon during tumor resection. The aim of my second project was the development of bioluminescent triglyceride probes for real-time non-invasive imaging of triglyceride uptake in vivo. Applying an optimized stategy for generating conjugates with luciferin, I developed several bioluminescent triglyceride probes. These imaging tools were validated in cells and mice and were shown to mimic the absorption of natural triglycerides. The developed probes enabled sensitive non-invasive real-time imaging and quantification of triglyceride absorption in living mice. Futhermore, these probes were successfully used to demonstrate the effects of the anti-obesity drug orlistat and the influence of the gut microbiota on the intestinal absorption of triglycerides in vivo. Considering the high importance of triglycerides for human health and nutrition and their implication in several pathologies such as obesity, metabolic syndrome, type 2 diabetes and cancer, the bioluminescent triglyceride probes could offer valuable imaging tools for preclinical research.