Aboveground net primary production provides valuable information on wildlife habitat, fire fuel loads, and forage availability. Aboveground net primary production in herbaceous plant communities is typically measured by clipping aboveground biomass. However, the high costs associated with physically harvesting plant biomass may prevent collecting sufficient data to account for natural spatial and temporal variability of vegetation at a landscape scale. Various double-sampling techniques have been developed to increase sample size while reducing cost. We applied a biomass estimation technique previously developed for estimating shrub biomass using representative samples or "reference units" to estimate herbaceous grassland biomass. Our reference units consisted of major grass species and functional groups that involved combining species with similar origin (native vs. introduced) and life-form characteristics. This study was conducted in 2010 on and around prairie dog colonies on the Buffalo Gap National Grassland in southwestern South Dakota, which provided a range of plant communities for testing the method. Results of the study demonstrate that reference units can provide accurate and precise estimates of herbaceous plant biomass on grasslands, including multi-species functional groups. Twenty-five of 26 double sampling calibrations were validated by time with no changes in observer estimation trends over the sampling season. Use of the reference unit method was consistent among observers and is a viable option for estimating and monitoring herbaceous grassland aboveground biomass.