Conventional and newly-developed techniques to determine the phosphorus (P) status of Lake Ontario phytoplankton were employed in September 2003, immediately after the passage of the storm system associated with Hurricane Isabel. Surface water (1–5 m) was collected at 29 stations, with selected stations sampled throughout the water column. Chemical estimates of total P concentrations were compared with proxies of P bioavailability: P enrichment bioassays of lake water, alkaline phosphatase activity (APA), and P-dependent bioreporter assays. Average total P (314 nM) and total chlorophyll-α (2.12 μg/L) concentrations measured in pelagic surface waters from throughout Lake Ontario suggest an oligotrophic status prevailed across much of this lake during the sample period. Autotrophic picoplankton (0.2–2 μm) displayed the highest growth rates and were grazed at the highest rate, whereas P-enrichment bioassays favored the production of autotrophic nanoplankton (2–20 μm) and autotrophic microplankton (> 20 μm) biomass. Average concentrations of bacteria (2.61 ×10¹⁰ cells/L) were higher than those measured during summer in a similar lake (Erie), whereas the average viral density (1.38 ×10¹⁰ virus particles/L) was similar. Pelagic stations exhibited higher APA than coastal stations; cyanobacterial bioreporter responses did not show high correlation with APA suggesting that proxies of P-demand based on residual effects (e.g., enzyme production) were not indicative of shorter-term biological responses related to planktonic growth (bioreporter genetic response). The combination of traditional chemical, biochemical (APA), and cutting-edge biological methods (bioreporter) provided information on nutrient concentrations and primary productivity throughout Lake Ontario, while concurrently allowing real-time assessment of P bioavailability.