Antimicrobial chemotherapeutic agents are widely used in poultry breeding to treat infection diseases. Their misuse contributes to the selection of resistant bacteria. There is great interest in non-invasive methods of monitoring and controlling the misuse of antimicrobials in poultry production for food safety purposes. Feather sampling could be a non-invasive way to monitor antimicrobial exposure in poultry through the analysis of feathers, in which residues persist. The objective of this study was to better understand the fate and distribution of oxytetracycline (OTC), sulfadiazine (SDZ) and trimethoprim (TMP) in feathers in order to better manage their monitoring in this matrix. In this article, the distribution of the three antimicrobials into feather over time was investigated through a longitudinal animal study on broiler chickens. During the 55-days study, feathers were sampled from the treated animals at 11 time points, segmented into nine barbs and rachis segments and analysed using a previously validated LC-MS/MS method. The results of analysed feathers segments showed that the distribution patterns within the feather seem to differ depending on the antimicrobial substance, the type of segment (rachis and barbs), but also on each segment over time. OTC, SDZ and TMP concentrations were higher in the whole barbs. Unlike OTC, SDZ and TMP appeared to have quite similar pattern of migration in the different feather segments. SDZ and TMP seems to migrate over time in a larger proportion into the upper barbs compared to OTC which have a relative equally distribution pattern over time. Although further experiments would be necessary to confirm the observed patterns, this exploratory study provides first general trend data on the proportions of SDZ, TMP and OTC in feathers segments over time. Feathers remain a promising matrix for the monitoring and surveillance and offer relevant information on the exposure of poultry to antimicrobial agents during their bird life.