We conducted a probability-based net tow sampling of Lake Superior in 2006 and compared the zooplankton biomass estimate with an estimate from laser optical plankton counter (LOPC) surveys. The net survey consisted of 52 sites stratified across three depth zones (0–30, 30–150, >150 m). The LOPC tow surveys were extensive and spatially covered much of Lake Superior (>1300 km of towing). The LOPC was field calibrated to Lake Superior zooplankton samples collected across the years of 2004 to 2006. The volume-weighted lakewide zooplankton biomass determined by traditional net tows to 100-m sample depth was 20.1 (±7.8 SD n = 52) mg dry-weight m^-3. The estimates varied by depth zones within the lake, where nearshore (0–30 m) estimates were highest and highly variable. Net sites for the LOPC field calibration were removed to allow for LOPC validation with independent nets; the resulting net-based estimate 20.0 (± 9.3 SD n = 38) mg dry-weight m^-3 and LOPC lake-wide estimate 19.1 (±3.3 SD) mg dry-weight m^-3 agreed well. Consistency across survey methods for lake-wide estimates suggested that LOPC survey data provides a comparable assessment tool to traditional nets for collecting zooplankton biomass data. We briefly compare our results with some observed historical patterns. Onshore—offshore trends in zooplankton biomass concentrations were similar to the last major lake-wide survey in 1973. The LOPC provided high resolution data on Zooplankton biomass distribution. Using simultaneously collected in situ sensor data, the LOPC zooplankton biomass distributions over horizontal and vertical space can be modeled as a function of temperature and fluorescence.