Fluorescent molecular-guided surgery (FGS) is at a tipping point in terms of clinical approval and adoption in a number cancer applications, with ongoing phase 0 and phase 1 clinical trials being carried out in a wide range of cancers using a wide range of agents. The pharmacokinetics of each of these agents and the physiology of these cancers can differ vastly on a patient-to-patient basis, bringing to question: how can one fairly compare different methodologies (defined as the combination of imaging agent, system, and protocol) and how can existing methodologies be further optimized? To this point, little methodology comparison has been carried out, and the majority of FGS optimization has concerned system development—on the level of maximizing signal-to-noise, dynamic detection range, and sensitivity—independently from traditional agent development—in terms of fluorophore brightness, toxicity, solubility, and binding affinity and specificity. Here we propose an inclusion of tumor and healthy tissue physiology (blood flow, vascular permeability, specific and nonspecific binding sites, extracellular matrix, interstitial pressure, etc.) variability into the optimization process and re-establish well-described task-based metrics for methodology optimization and comparing quality of one methodology to another. Two salient conclusions were identified: (1) contrast-tobackground variability is a simple metric that correlates with difficult-to-carry-out task-based metrics for comparing methodologies, and (2) paired-agent imaging protocols offer unique advantages over singleimaging- agent studies for mitigating confounding tumor and background physiology variability.