Western blotting with fluorescence detection offers the possibility of detecting multiple targets simultaneously on a single blot. Primary antibodies are increasingly available from multiple hosts, and there are now a wide variety of dye labels to exploit multiple imaging channels. If primary and secondary antibodies are selected so that individual targets can be discriminated, multiple antigens can be detected and quantified in a single experiment. Current fluorescence imaging instrumentation offers multiple detection channels and gives sensitivity comparable to other methods. The method described in this article allows multiple targets to be quantified simultaneously and reduces the need for stripping and re-probing. It also allows loading controls to be detected alongside the targets of interest. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Five-plex western blot detection, including tubulin detection for loading control.