Identifying pro-inflammatory macrophages (M1) is of immense importance to diagnose, monitor, and treat various pathologies. In addition, adoptive cell therapies, where harvested cells are isolated, modified to express an M1-like phenotype, then re-implanted to the patient, are also becoming more prevalent to treat diseases such as cancer. In a step toward identifying, labeling, and monitoring macrophage phenotype for adoptive cell therapies, we developed a reactive oxygen species (ROS)-sensitive, gold nanoparticle (AuNP) that fluorescently labels M1 macrophages. AuNPs are electrostatically coated with a proteolysis resistant, fluorescein isothiocyanate-conjugated, poly-D-lysine (PDL-FITC) that is susceptible to backbone cleavage by ROS. When PDL-FITC is bound to AuNPs, fluorescence is quenched via a combination of nanoparticle surface (NSET) and Forster resonance (FRET) energy transfer mechanisms. Upon ROS-induced cleavage of PDL-FITC, up to a 7-fold change in fluorescence is demonstrated. PDL-FITC AuNPs were loaded into RAW 264.7 macrophages (RAWs) and primary bone marrow- derived macrophages (BMDMs) prior to in vitro polarization. For both cell types, detectable differences in intracellular fluorescence were observed between M1 polarized and non-stimulated (M0) control groups after 24 h using both confocal imaging and flow cytometry. PDL-FITC AuNPs can potentially be useful in identifying M1 macrophages within diverse cell populations and provide longitudinal macrophage response data to external cues.