This USGS Data Release represents the synthetic and real data from hydrologically diverse streams used to test the performance and limitations of the Two-tracer Ratio-based Mixing Model (TRaMM) which uses high-frequency measures of two tracers (A and B) and streamflow to separate total streamflow into water from slowflow and fastflow sources. The ratio between the concentrations of the two tracers is used to create a time-variable estimate of the concentration of each tracer in the fastflow end-member. Synthetic data from a groundwater dominated stream and an overland flow dominated stream were used to test the sensitivity of the model to various conditions and tracer concentrations. The sensitivity analysis provides understanding of the relation between the inputs and outputs of the model while providing information regarding its practicality and limitations. High-frequency nitrate and specific conductance data from Chesterville Branch, Maryland and Indian Creek, Kansas in 2013 were used to test the model under real-world conditions. These data support the following publication: Kronholm, S.C. and Capel, P.D., 2016, Estimation of time-variable fast flow path chemical concentrations for application in tracer-based hydrograph separation analyses: Water Resour. Res., 52, 6881-6896, https://doi.org/10.1002/2016WR018797.