Most animals establish long-term symbiotic associations with bacteria that are critical for normal host physiology. The symbiosis that forms between the Hawaiian squidEuprymna scolopesand the bioluminescent bacteriumVibrio fischeriserves as an important model system for investigating the molecular mechanisms that promote animal-bacterial symbioses.E.scolopeshatch from their eggs uncolonized, which has led to the development of squid-colonization assays that are based on introducing culture-grownV.fischericells to freshly hatched juvenile squid. Recent studies have revealed that strains often exhibit large differences in how they establish symbiosis. Therefore, we sought to develop a simplified and reproducible protocol that permits researchers to determine appropriate inoculum levels and provides a platform to standardize the assay across different laboratories. In our protocol, we adapt a method commonly used for evaluating the infectivity of pathogens to quantify the symbiotic capacity ofV.fischeristrains. The resulting metric, the symbiotic dose-50 (SD50), estimates the inoculum level that is necessary for a specificV.fischeristrain to establish a light-emitting symbiosis. Relative to other protocols, our method requires 2–5-fold fewer animals. Furthermore, the power analysis presented here suggests that the protocol can detect up to a 3-fold change in the SD50between different strains.