The Caco-2 cell line is commonly used as a model to study various events occuring at the intestinal level such as the physiological impact of toxins or the absorption of nutriments. More recently, co-cultures integrating enterocyte-like and goblet cells types, namely Caco-2 and HT29-MTX cells, have been reported as promising models of a tunable1 and functional 2 epithelial barrier. One interest of including HT29-MTX cells is their ability to secrete mucins. It is therefore expected that the cell culture will be lined by a mucus layer, with functional consequences on absorption or bacterial adhesion for example. However, the spatial characteristics of this mucus layer (e.g. distribution or volume) is not fully described. The objective of this work was to visualize mucins in a co-culture of Caco-2/HT29-MTX cells, and to set up a method of mucus characterization based on image analysis. Caco-2 and HT29-MTX cells were routinely grown in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were seeded at a density of 2.105 cells/cm2 on transwells in 24-well culture plates, at a ratio of 9:1 (Caco-2:HT29-MTX). Incubation was performed in a humidified atmosphere of 5% CO2 at 37 °C. On day 21, cultures were fixed with 4% PFA and double-stained for F-actin using phalloidin-rhodamine, and for sialic acid using WGA-Alexa488. This aimed at detecting the cytoskeleton (thereby enabling fine contouring of each cell) and mucins, respectively. Images were acquired on a Zeiss-LSM 880 confocal microscope. Two independent culture wells were observed. Five 3-D images (consisting of around 80 stacked images on average) were acquired per well, resulting in a dataset of 10 images. From a qualitative point of view, it was observed that cells at day 21 formed mostly a monolayer. Rather than being flat, cultures showed a clear topographic pattern, with domes that could reach 135 µm in height. Mucins were detected mainly on the apical side of the cells. Brightly-stained mucin clusters were visible in the extracellular apical space in close vicinity of some cells, most likely HT29-MTX cells. In addition, a more diffuse signal was also observed, sometimes lining large parts of the observation sites. This may correspond to secreted mucins spreading on top of the culture to form a typical intestinal mucus. With the objective of eventually study the impact of a treatment (e.g. digested food constituents) on this mucus structure, a method of image analysis was developed. Using the open-source software ilastik3, pixels were classified into three categories corresponding to bright staining mucin clusters, diffuse mucin staining and background. This analysis provides quantitative measurements on average mucin intensity and the volume proportion and average heights of mucin clusters.