A simple and sensitive chromatographic method has been developed for the quantitative analysis of an antiviral agent, daclatasvir (DCV), that commonly prescribed for the treatment of hepatitis C viral (HCV) infection. The method was applied to detect DCV in human plasma and real blood samples collected from patients diagnosed with HCV and treated with DCV. The analysis strategy was based on recording the native fluorescence of DCV in plasma, after pre-column treatment to precipitate the plasma proteins using a readily applicable protocol. Chromatographic conductions, factors influencing the fluorescence and stability studies were also investigated. Furthermore, the method was validated according to the International Conference on Harmonization (ICH) guidelines and could be used to detect DCV in plasma over a linear range of 1.0-4000 ng/mL, with an acceptable sensitivity as the limit of detection (LOD) was 0.025 ng/mL. In addition, the study was extended to evaluate the pharmacokinetic interaction between DCV and a co-prescribed antidepressant drug, fluoxetine (FLX) in real blood samples, collected from volunteering patients who were diagnosed with HCV and treated with DCV alone or combined with FLX. The results showed a significant influence of FLX on the pharmacokinetic profile of DCV. The findings observed in this study could be used by clinical pharmacists to adjust the DCV dose, when combined with FLX, during the HCV treatment.