Although rotifers are important components of aquatic food webs and suitable sampling methods have been described and tested in the peer-reviewed literature, they are frequently overlooked or quantified with improper methods (e.g., mesh sizes ≥ 63 µm) in freshwater ecology studies. As a result, we believe that the role of rotifers in aquatic food webs and ecosystem processes remains underappreciated, and this conceptual shortfall is exacerbated by the continued use of improper sampling methodology. We examined density and biomass estimates of metazoan zooplankton in the Upper Mississippi and Missouri rivers from two sampling methods. The macrozooplankton method was designed to target cladocerans, adult and juvenile copepods, by filtering 180 L water through a 63-µm mesh. The microzooplankton method was designed to target rotifers and copepod nauplii by filtering 18 L water through a 20-µm mesh. The macrozooplankton method underestimated the density and biomass of common rotifers by two to three orders of magnitude, a far greater amount of error than reported in previous studies. This bias in density estimates for rotifers decreased with increasing mean length of rotifers. The microzooplankton method proved to be ineffective for quantifying cladoceran species richness or cladoceran density at the species level. We urge aquatic ecologists to match their sampling methodology with the specific goals of their study. An accurate understanding of the role of rotifers in freshwater ecosystems will only be possible when the use of appropriate methodology becomes the rule rather than the exception.