Florida’s unique aquatic landscape, with 7,783 natural lakes, features predominantly shallow lakes formed by limestone dissolution, creating distinctive limnological traits. Despite this uniqueness, research on lacustrine food webs in Florida is limited and mostly focuses on economically important lakes. Furthermore, the existing studies on dietary linkages focus largely on fish, underrepresenting lower trophic level organisms and potentially overlooking critical food web interactions. Additionally, the use of carbon isotopes for diet tracing in these shallow and productive solution lakes, which lack profundal habitats found in northern ecosystems, has shown mixed success. Thus, we aim to fill knowledge gaps in food web dynamics in Florida’s unique subtropical lakes, particularly the utility of carbon isotopes and signatures in lower trophic level organisms (i.e., plankton, macroinvertebrates, and macrophytes). In November 2024, we conducted a comprehensive, multi-method survey of the hypereutrophic Lochloosa Lake in northcentral Florida, USA, to capture its ecological complexity. Collected organisms are being identified and sorted based on ecological niche, and tissue samples are being processed for stable carbon and nitrogen isotope values (δ13C and δ15N). If carbon isotope tracers differentiate, we anticipate Bayesian modeling to determine plankton as the most common dietary endpoint for pelagic fish, and a mixture of macrophytes and plankton for littoral forage fish. If carbon isotopes don’t differentiate, additional isotope tracers (i.e., sulfur or CSIA) may be incorporated. Based on field observations, the highest trophic positions will be dominated by Micropterus salmoides and Lepisosteus platyrhincus. Our study will provide insight into trophic interactions in highly productive, subtropical lakes, with implications for future conservation and management of Florida’s freshwater ecosystems. More specifically, our study will create novel baseline data on lentic food webs, supporting future research on the impact of climatic change and/or invasive species on lacustrine ecosystems in Florida.