Drug discovery for central nervous system (CNS) targets is a high stakes process with estimated success rates below ten percent. Dose scaling, penetration through the blood-brain-barrier (BBB), and potency are among the various challenges involved in developing drugs for CNS targets. The standard approach to evaluate some of these parameters is dosing lead therapeutic compounds via intravenous delivery and assessing their brain levels via tissue homogenization and ex vivo quantification. Although a cost and time effective approach, brain homogenization lacks pharmacokinetic spatial resolution and normalizes drug levels to the entire brain volume. The brain, however, is known to have regional differences in cellular composition, transporters, BBB permeability, and drug-metabolizing enzymes, factors that could significantly affect pharmacological assessments during drug discovery. In this study we employ electrochemical aptamer-based sensors, a technology that allows in situ, real-time molecular monitoring in live animals, to reveal significant differences in the pharmacokinetics of drug uptake and accumulation in the brain of mice. Using vancomycin in the context of penetrating brain injury (PBI), our results highlight that potency may be significantly affected by PBI location. Additionally, more accurate dose scaling and delivery for deep brain wounds could be achieved by adjusting route of administration based on real-time-measured pharmacokinetic profiles, for example by changing delivery from intravenous to intracerebroventricular dosing. We emphasize the issue of establishing accurate pharmacological parameters during preclinical drug discovery efforts and underline the value of aptamer-based sensors for precise estimations of drug pharmacokinetics, transport across the BBB, and effective dose delivery during preclinical trials.