Background/Objectives: The aim of this study was to determine the plasma and muscle pharmacokinetics of ceftriaxone (25 mg/kg) in tilapia after different administration routes. Methods: Two hundred and sixteen fish maintained at 30 ± 1.5 °C were divided equally into three treatment groups: intravascular (IV), intraperitoneal (IP), and intramuscular (IM). Ceftriaxone concentrations were quantified using high-performance liquid chromatography, and pharmacokinetic parameters were calculated by non-compartmental analysis. Results: The plasma total body clearance, volume of distribution at steady state, and elimination half-life (t1/2λz) were 0.22 L/h/kg, 0.85 L/kg, and 5.27 h, respectively. The t1/2λz values were comparable among the IV, IP, and IM injection groups. The peak plasma concentration was 37.71 ± 3.12 µg/mL and 40.51 ± 2.77 µg/mL following IP and IM injection, respectively. The bioavailability was 67.04% for IP and 101.48% for IM. The peak muscle concentration was 9.49 ± 0.75 µg/g for IV, 5.71 ± 0.85 µg/g for IP, and 12.24 ± 2.41 µg/g for IM injection. The AUC0–∞muscle/AUC0–∞plasma ratio was 0.23, 0.18, and 0.30 for the IV, IP, and IM groups, respectively. The AUCmuscle/AUCplasma indicates the ratio of drug penetration into the muscle, and a value less than 1 indicates that ceftriaxone penetrates into muscle tissue at a low ratio. Conclusions: These results indicate that ceftriaxone is well absorbed after IP and IM injections and passes into muscle tissue at a low tissue penetration. Ceftriaxone can be administered via IP and IM injection in Nile tilapia; nevertheless, its therapeutic efficacy requires evaluation.