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While particle carriers have potential to revolutionize disease treatment, using these carriers requires knowledge of spatial and temporal biodistribution. The goal of this study was to track orally administered particle uptake and trafficking through the murine gastrointestinal (GI) tract using multispectral optoacoustic tomography (MSOT). Polylactic acid (PLA) particles encapsulating AlexaFluor 680 (AF680) dye conjugated to bovine serum albumin (BSA) were orally gavaged into mice. Particle uptake and trafficking were observed using MSOT imaging with subsequent confirmation of particle uptake via fluorescent microscopy. Mice treated with PLA-AF680-BSA particles exhibited MSOT signal within the small bowel wall at 1 and 6 h, colon wall at 6, 12, and 24 h, and mesenteric lymph node 24 and 48 h. Particle localization identified using MSOT correlated with fluorescence microscopy. Despite the potential of GI tract motion artifacts, MSOT allowed for teal-time tracking of particles within the GI tract in a non-invasive and real-time manner. Future use of MSOT in conjunction with particles containing both protein-conjugated fluorophores as well as therapeutic agents could allow for non-invasive, real time tracking of particle uptake and drug delivery.