Electrochemical aptamer-based (EAB) sensors utilize the binding-induced conformational change of an electrode-attached, redox-reporter-modified aptamer to transduce target recognition into an easily measurable electrochemical output. Because this binding-induced signal transduction mechanism is independent of the chemical reactivity of the targets, EAB sensors are generalizable to a wide range of clinically and scientifically important molecular targets, including metabolites, drugs, hormones, and protein biomarkers. This signal transduction mechanism also renders EAB sensors selective enough to perform such measurements in complex sample matrices, such as undiluted saliva, foodstuffs, urine, and whole blood. Indeed, EAB sensors even support real-time, seconds-resolved molecular monitoring in situ in the living body, an ability that, in turn, supports such advances as closed-loop, feed-back controlled drug delivery. As the first chapter of my thesis work, I developed a more systematic means of re-engineering aptamers to undergo the binding-induced folding required for them to function in EAB sensors. Specifically, I used circular dichroism to identify truncation variants of established aptamers against methotrexate, tryptophan, and cocaine that undergo a large-scale binding-induced conformational change suitable for use in EAB sensors. For example, using circular dichroism to monitor for binding-induced conformational changes, I identified an intermediate length construct of the tryptophan aptamer that, when adapted into an EAB sensor, produces a large change in electrochemical signal over physiological tryptophan concentrations. Following this same procedure, I next re-engineered methotrexate and cocaine aptamers for adaptation into similarly high-performance EAB sensors, ultimately supporting clinically relevant measurements of both drugs in situ in the living body. In my second thesis chapter, I explored improvements in the fabrication of EAB sensors. Historically, EAB sensors have been fabricated by sequentially treating their gold electrode in a solution of thiol-modified aptamer followed by a second incubation in dilute mercaptohexanol, which “backfills” the space between aptamers to form a continuous, well-packed monolayer. In contrast to this sequential method, there have been fewer common reports of a “codeposition” method that employs single-step incubation in a mixture of mercaptohexanol and thiol-modified aptamer. Prior to my work, however, there had been no direct comparison of the performance of EAB sensors fabricated using these two distinct approaches. Performing such a comparison, I discovered that, in addition to being more convenient, codeposition also often leads to improved EAB sensor performance. With a high-performance tryptophan EAB sensor in hand, for the final chapter of my thesis I characterized the metabolism of tryptophan, an essential amino acid that is not only a building block for protein synthesis but also a precursor for the biosynthesis of many co-enzymes and neuromodulators, in situ in the bodies of live rats. Specifically, I collected high-frequency, real-time measurements of plasma free tryptophan in studies that unveiled new insights into the regulation of tryptophan metabolism. In doing so, I found that, upon challenge with a single, high tryptophan dose, the resulting return-to-homeostasis kinetics are well-described by a Michaelis-Menten model limited by the Michaelis constant of tryptophan dioxygenase, the enzyme known to account for most of the degradation of tryptophan in mammals. And upon a series of sequential tryptophan injections, the rate with which plasma free tryptophan concentrations return to homeostasis accelerates, an observation that is consistent with reported literature that the activity of tryptophan dioxygenase in the liver is increased upon tryptophan challenge. Despite this increase in tryptophan degradation rate, the plasma free tryptophan concentrations always returned to the same pre-challenge baseline, suggesting additional control of plasma tryptophan level via regulation of the amino acid’s mobilization from tissue reserves.