In this study we tested a glucuronide-based micro-PET probe for the in vivo detection of βG-expressing tumors in a mouse model. To achieve specific imaging of βG-expressing tumors, we developed a prcrobe for micro-PET by labeling 124iodine to hydrophilic phenolphthalein glucuronide (PTH-G) to form a 124I-phenolphthalein glucuronide probe (124I-PTHG). Enzymatic hydrolysis was anticipated to convert the probe into water-insoluble 124Iphenolphthalein (124I-PTH) for preferential retention at sites displaying βG activity. 124I-PTH-G was selectively converted to 124I-PTH, which accumulated at βG-expressing CT26 (CT26/ βG) cells and tumors in vitro. When 124I-PTH-G was intravenously injected into mice bearing both CT26 and CT26/ βG tumors, the micro-PET images produced by the converted 124IPTH clearly showed the locations of CT26/ βG tumors. Autoradiographic and biodistribution analyses ot the probe showed that the in vivo accumulation of radioactive signals were 3.6, 3.4 and 3.3-fold higher in the CT26/ βG tumors than parental CT26 tumors at 1 h, 3 h and 20 h, respectively, after injection of the probe, These results demonstrate that the hydrophilic-hydrophobic conversion of the 124IPTH-G probe can aid in the imaging of βG-expressing tumors in vivo. The targeting strategy described in this report may provide a valuable tool to diagnose βG activity in vivo to ersonalize glucuronide prodrug targeted therapy.