Incorporation of radioactive functionality into nanoparticles (NPs) is an emerging strategy to quantitatively evaluate their in vivo performance with quantitative biodistribution and imaging modalities such as positron emission tomography (PET). Single photon emission computed tomography (SPECT) has the ability to detect a range of photonic energies, and therefore can be employed in multispectral imaging using multiple radionuclides. When properly integrated with NP probes, this independent nuclide tracking can be used to characterize important in vivo parameters such as radiolabeling stability, surface anchor stability, and biological parameters such as enzyme activity. In this study, a multifunctional NP agent was designed to passively target tumors and characterize matrix metalloproteinase (MMP) activity using a dual-radiolabeling strategy, which involves the synthesis of an imaging agent containing two distinct radionuclides, separated by a cleavable linker, whose gamma emissions can be spectrally differentiated. Specifically, the surfaces of gold nanoparticles were functionalized with a peptide containing a tyrosine residue to radiolabel with I-125 and a DTPA chelator to radiolabel with radiometals, which are separated by a sequence cleaved specifically in the presence of MMP9. Peptide cleavage from the nanoparticle probe by MMP9 was observed in vitro, and in vivo spectroscopic SPECT imaging was performed with the dual-radiolabeled probe in tumor-bearing mice. I-125 and In-111 could be independently tracked in vivo, and distinct pharmacokinetic profiles of the probes were observed in tumors with either high or low MMP9 expression, a result that shows the promising ability of these radiolabeled nanoparticles to differentiate between tumors with differing MMP9 expressions.