N‐acetyl‐l‐cysteine (NAC) has been suggested as an antioxidant that can alleviate the negative effects of stress conditions in broilers. However, knowledge of its pharmacokinetics (PK) in this avian species is very limited. Therefore, the study aimed to shed more light on the PK properties of NAC in chickens. Broilers were subjected to single intravenous (i.v.) or oral (p.o.) treatment or multiple NAC administrations via the feed. Drug concentrations were determined by LC–MS/MS, and the data were subjected to non‐compartmental analysis and modeled by non‐linear mixed effect approach. NAC was eliminated in a short time after i.v. treatment, with a t1/2el of 0.93 (0.59–2.09) h. It showed limited distribution with population mean of volumes of distribution in the central and peripheral compartments V1 of 0.148 L/kg and V2 of 0.199 L/kg, respectively, and Vdarea of 0.39 (0.258–0.635) L/kg. The value of MRT was 1.76 h (range of 0.96–2.69, p < .05) after single p.o. treatment, indicating a twofold increase if compared to i.v. administration (0.87 h, 0.55–1.78). Both methods of Pk analysis revealed very limited bioavailability, <10%. Feeding behavior led to a later achievement of lower maximum plasma concentrations (5.74, range of 3.44–9.32 μg/mL, p < .05), which were maintained during the 5 days of treatment.