Enrofloxacin (ENR) has been widely used to treat bacterial infections in catfish aquaculture. However, little is known about the elimination of ENR in fish following treatment in aquaculture ponds. The aim of this study was through on-farm trials to establish the withdrawal period for ENR following treatment of striped catfish (Pangasianodon hypophthalmus). Three catfish ponds were treated daily for five consecutive days with medicated pelleted feed containing ENR (10mg/kg body weight) following normal farmer procedures. Sampling of catfish muscle/skin for residue analysis was done one day before the first feeding with medicated feed as well as three times during and five times after (7, 15, 30, 45, and 200days) application of medicated feed. ENR and its metabolite ciprofloxacin (CIP) residues were analyzed by Liquid Chromatography coupled to Mass Spectrometry (LC-MS/MS). As expected, ENR and CIP residues initially accumulated in mixed muscle/skin and the concentrations were subsequently reduced during the sampling period. After 45days (D45), the concentration of total ENR and CIP was 30.8±4.1μg/kg. Our results show that a withdrawal time of 45days is sufficient for striped catfish treated with ENR, i.e. below the Maximum Residue Limit (MRL) assigned by the Commission of the European Communities (100μg/kg) in fish for human consumption but above the action level proposed by the US Food and Drug Administration (FDA) (5μg/kg). Further, the 30-days withdraw period declared on the ENR products sold to catfish farmers is inadequate. The ENR and CIP residues found in skin about 200days after treatment warrants follow-up studies, e.g. of possible food safety risks. •An on-farm trial with striped catfish in Vietnam to establish the withdrawal period for enrofloxacin showed that 45days after treatment fish muscle contained a total of 30.8±4.1μg/kg ENR and CIP.•A withdraw time of 45days meets the Maximum Residue Limit (MRL) assigned by the Commission of the European Communities (100μg/kg) in fish for human consumption, but is above the action level proposed by the Food and Drug Administration (FDA) (5μg/kg) in the United States.•The 30-days withdraw period declared by the manufacturers of ENR products sold to catfish farmers is inadequate.•At harvest, skin samples still contained high levels of ENR and CIP metabolites which may constitute a food safety risk when catfish skin-derivate products are sold for human consumption.