The current study aimed to explore the pharmacokinetics of danofloxacin in non‐laying hens after a single oral (PO) and intravenous (IV) dose, both at 5 mg/kg body weight (BW). Eighteen 13‐week‐old healthy hens were equally and randomly divided into two groups. After both doses, blood samples (approximately 1 ml) were collected at different time points. Danofloxacin concentrations were quantified by a validated high‐performance liquid chromatography (HPLC) method followed by a non‐compartmental analysis using the software of WinNonLin. The elimination half‐lives (t1/2λzs) after PO and IV routes were determined as 8.15 ± 3.37 and 7.69 ± 3.40 h, respectively. After IV administration, danofloxacin had an initial concentration (C0) of 3.62 μg/ml, a volume of distribution at steady state (VSS) of 3579.72 ± 454.29 ml/kg, and a total body clearance (Cl) of 0.49 ml/h/g. After PO administration, the absolute bioavailability and absorption half‐life (t1/2ka) were calculated as 100.99% ± 23.10% and 0.82 ± 0.58 h, respectively. Based on the calculated ratio values of AUC/MIC and Cmax/MIC, an oral dose of 5 mg/kg danofloxacin would be expected to successfully treat hens infected with strains with MIC values ≤0.1 μg/ml.