We developed an improved method for sensitive and selective Trp sensing via the application of an optimized multi-scan rate sawhorse waveform using FSCV at CFMs. We demonstrated four-fold higher sensitivity for Trp sensing over previous methods and a six-fold higher current density for Trp signals than those for Tyr, one of the primary interfering species that has similar oxidation potentials. We utilized the optimized method and measured exogenously applied extracellular Trp in PC-12 and PC-12 with overexpression of TPH2. The time-dependent measurements revealed similar increase, but interesting differences in the decay curves. We also demonstrated the utility of the method in measuring time resolved endogenous Trp dynamics in pinealocytes either exposed to water or water with norepinephrine. There were clear differences between the two groups. Altogether, our optimized method has the spatiotemporal resolution, sensitivity, and selectivity to monitor exogenously added and endogenously present Trp dynamics in different cell cultures. These proof-of-concept studies are promising for anticipated future work in vivo.