Flavobacterium columnare, the causative agent of columnaris disease, is a significant pathogen of many freshwater fish species worldwide and is considered one of the most important pathogens impacting the channel catfish (Ictalurus punctatus Rafinesque) industry in the United States. Recent research has demonstrated that F. columnare exhibits a chemotactic response to mucus from the skin of catfish and this response may be important for pathogenesis. In this study, a culture independent method was developed to quantify the chemotactic response of F. columnare to skin mucus. The method employs the use of blind-well chemotaxis chambers which overcomes difficulties using the traditional capillary tube assay and uses a cell proliferation assay to quantify viable cells which reduces the time and labor associated with culturing the bacterium. Application of the method to two sets of catfish skin mucus samples demonstrated that there is variation in the chemotactic response of F. columnare to individual catfish mucus samples, and similar results were obtained to previously published research using the traditional capillary tube method. The use of this method will enhance the ability to further characterize the chemotactic response of F. columnare to catfish skin mucus in order to increase the understanding of this important host-pathogen interaction.