Photoacoustic (PA) imaging is a developing diagnostic technique where multiple species can be simultaneously imaged with high spatial resolution in 3D if the absorbance spectrum of each species is distinct and separable. However, multiplexed PA imaging has been greatly limited by the availability of spectrally separable contrast agents that can be used in vivo. Toward this end, we present the formation and application of a series of poly ethylene glycol (PEG)-coated nanoparticles (NPs) with unique separable absorbance profiles suitable for simultaneous multiplexed imaging. As a proof-of-concept, we demonstrate this form of mixed-sample multiplexed imaging, using cRGD peptide surface-modified NPs with nonmodified NPs in a murine subcutaneous Lewis lung carcinoma tumor model. The simultaneous imaging of nonmodified NPs provides an “internal standard”, to deconvolute the contributions of active-ligand and passive-NP targeting effects. Particles with 25% surface cRGD modification display 52 ± 22 fold higher liver to tumor ratio accumulation levels, while the same set of particles display only 9.8 ± 4 fold accumulation levels when internally normalized. The pharmacokinetic profiles of targeted and nontargeted NPs can be simultaneously tracked in real-time to study how biodistribtions of particles are affected by ligand modification. The internal normalization of control particles greatly enhances the precision and decreases the number of animals needed in studies of nanoparticle targeting. These new dyes are an enabling technology for PA imaging of NP fate and targeting. This is the first demonstration of real-time multiplexed PA imaging of mixed-targeted samples in vivo.