We quantify the efficacy of flow-through nanohole sensing, as compared to the established flow-over format, through scaling analysis and numerical simulation. Nanohole arrays represent a growing niche within surface plasmon resonance-based sensing methods, and employing the nanoholes as nanochannels can enhance transport and analytical response. The additional benefit offered by flow-through operation is, however, a complex function of operating parameters and application-specific binding chemistry. Compared here are flow-over sensors and flow-through nanohole array sensors with equivalent sensing area, where the nanohole array sensing area is taken as the inner-walls of the nanoholes. The footprints of the sensors are similar (e.g., a square 20 μm wide flow-over sensor has an equivalent sensing area as a square 30 μm wide array of 300 nm diameter nanoholes with 450 nm periodicity in a 100 nm thick gold film). Considering transport alone, an analysis here shows that given equivalent sensing area and flow rate the flow-through nanohole format enables greatly increased flux of analytes to the sensing surface (e.g., 40-fold for the case of Q = 10 nL/min). Including both transport and binding kinetics, a computational model, validated by experimental data, provides guidelines for performance as a function of binding time constant, analyte diffusivity, and running parameters. For common binding kinetics and analytes, flow-through nanohole arrays offer ∼10-fold improvement in response time, with a maximum of 20-fold improvement for small biomolecules with rapid kinetics.