The net anthropogenic nitrogen input (NANI) approach is a simple quasi-mass-balance that estimates the human-induced nitrogen inputs to a watershed. Across a wide range of watersheds, NANI has been shown to be a good predictor of riverine nitrogen export. In this paper, we review various methodologies proposed for NANI estimation since its first introduction and evaluate alternative calculations suggested by previous literature. Our work is the first study in which a consistent NANI calculation method is applied across the U.S. watersheds and tested against available riverine N flux estimates. Among the tested methodologies, yield-based estimation of agricultural N fixation (instead of crop area-based) made the largest difference, especially in some Mississippi watersheds where the tile drainage was a significant factor reducing watershed N retention. Across the U.S. watersheds, NANI was particularly sensitive to farm N fertilizer application, cattle N consumption, N fixation by soybeans and alfalfa, and N yield by corn, soybeans, and pasture, although their relative importance varied among different regions.