Autophagy is a key biological process that has proven extremely difficult to detect noninvasively. To address this, an autophagy detecting nanoparticle (ADN) was recently developed, consisting of an iron oxide nanoparticle decorated with cathepsin-cleavable arginine-rich peptides bound to the near-infrared fluorochrome Cy5.5. Activation of the probe in autophagolysosomes results in the emission of Cy5.5 fluorescence and provides a measure of autophagosome flux. However, in the early autophagosome ADN fluorescence is silent due to fluorochrome stacking. Here, we introduce to ADN a second non-cleavable fluorophore that allows the probe to be tracked through all stages of autophagy. The nature of the secondary/tracking fluorophore has a profound effect on the activation of ADN and the emission of Cy5.5 fluorescence. The lead candidate, ADN2 (featuring AZDye546 as the secondary fluorophore) has the highest activation rate and change in Cy5.5 fluorescence. Absorbance and fluorescence spectrophotometry methods show that the negatively charged AZDye546 interacts with the positively charged polyarginine motifs of the Cy5.5-polyArg activatable fluorophore, resulting in enhanced baseline quenching of the Cy5.5 signal in the nanoprobe. Flow cytometry shows that the activation of ADN2 remains specific for autophagy and is strongly modulated by classical regulators of autophagy (starvation, bafilomycin) and genetic deletion of key autophagy proteins (ATG5, ATG7). ADN2 co-localized strongly with LC3-GFP positive autophagosomes and provided readouts of in vivo probe delivery and activation in the hearts of fed/starved mice. ADN2 enhances the ability to image autophagy without genetic transfection of cells/animals and underscores the possible effects for unanticipated interactions between fluorochromes and other moieties on the surface of decorated nanoparticles.