The melanocortin 1 receptor (MC1R) is overexpressed in most melanoma metastases, making it a promising target for imaging of melanomas. In this study, the expression of MC1R in a large fraction of patients with melanoma was confirmed using mRNA and tissue microarray. Here, we have characterized the in vivo tumor and tissue distribution and pharmacokinetics (PK) of uptake and clearance of a MC1R specific peptidomimetic ligand conjugated to a near-infrared fluorescent dye. We propose an interdisciplinary framework to bridge the different time and space scales of ligand-tumor-host interactions: intravital fluorescence microscopy to quantify probe internalization at the cellular level, a xenograft tumor model for whole body pharmacokinetics, and a computational pharmacokinetic model for integration and interpretation of experimental data. Administration of the probe into mice bearing tumors with high and low MC1R expression demonstrated normalized image intensities that correlated with expression levels (p < 0.05). The biodistribution study showed high kidney uptake as early as 30 min postinjection. The PK computational model predicted the presence of receptors in the kidneys with a lower affinity, but at higher numbers than in the tumors. As the mouse kidney is known to express the MC5R, this hypothesis was confirmed by both coinjection of a ligand with higher MC5R affinity compared to MC1R and by injection of lower probe concentrations (e.g., 1 nmol/kg), both leading to decreased kidney accumulation of the MC1R ligand. In addition, through this interdisciplinary approach we could predict the rates of ligand accumulation and clearance into and from organs and tumors, and the amount of injected ligand required to have maximum specific retention in tumors. These predictions have potential to aid in the translation of a targeted agent from lab to the clinic. In conclusion, the characterized MC1R-specific probe has excellent potential for in vivo detection of melanoma metastases. The process of cell-surface marker validation, targeted imaging probe development, and in vitro, in vivo, and in silico characterization described in this study can be generally applied to preclinical development of targeted agents.