Here, we describe a method that detects bacterial binding to mucus on slot-blot membranes. Mucins, the large, highly glycosylated proteins forming the mucus gel, attach slower to microwell plates than the small less glycosylated components of mucus, which quickly coat plastic surfaces and thereby limit mucin attachment. Therefore, other methods are necessary to retain significant levels of mucins when performing assays on mixed samples. In the method described here, samples are dot-blotted onto a polyvinylidene fluoride membrane and incubated with biotinylated bacteria. Bacterial adhesion to mucins is determined after visualization of the slot-blots with an infrared imaging system and quantification of the image pixel intensities. This method is also useful for the detection of bacterial binding to enzyme-treated mucins, for example with sialidase, and binding intensities can be compared to nonenzyme (mock)-treated mucins.