The validation of high sensitivity and high resolution microSPECT/CT imaging for tracking the in vivo pathway and fate of an ¹¹¹Indium-labeled (¹¹¹In) amphiphilic diblock copolymer micelle formulation is presented. Heterobifunctional poly(ethylene glycol) was used to initiate cationic ring opening polymerization of ε-caprolactone, which was then conjugated to 2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (p-SCN-Bn-DTPA) for chelation with ¹¹¹In. The micelles were characterized in terms of their physicochemical properties including size, size distribution, zeta-potential, and radiochemical purity. Elimination kinetics and tissue deposition were evaluated in healthy mice following administration of ¹¹¹In-micelles, ¹¹¹In-DTPA-b-PCL unimers (i.e., administered under the critical micelle concentration) or ¹¹¹In-Bn-DTPA. Healthy and MDA-MB-231 tumor-bearing mice were imaged using microSPECT/CT following iv administration of ¹¹¹In-micelles or ¹¹¹In-Bn-DTPA. Overall, incorporation of ¹¹¹In onto the surface of thermodynamically stable micelles results in long plasma residence times for the radionuclide and preferential localization within the spleen (22 ± 5% i.d./g), liver (13 ± 3% i.d./g), and tumor (9 ± 2% i.d./g). MicroSPECT/CT imaging provided noninvasive longitudinal visualization of circulation dynamics and tissue deposition. A strong correlation between image-based region of interest (ROI) analysis and biodistribution data was found, implying that nuclear imaging can be used as a noninvasive tool to accurately quantify tissue distribution. As well, the image-based assessment provided unique insight into the intratumoral distribution of the micelles in vivo.