In the previously published part of this study, we detailed a novel strategy based on dispersive liquid-liquid microextraction to extract and preconcentrate nine fluoroquinolones in porcine blood. Moreover, we presented the optimized experimental conditions to obtain complete CE separation between target analytes. Consequently, this second part reports the validation of the developed method to determine flumenique, difloxacin, enrofloxacin, marbofloxacin, ofloxacin, ciprofloxacin, through univariate calibration, and enoxacin, danofloxacin, and gatifloxacin through multivariate curve resolution analysis. The validation was performed according to FDA guidelines for bioanalytical assay procedures and the European Directive 2002/657 to demonstrate that the results are reliable. The method was applied for the determination of fluoroquinolones in real samples. Results indicated a high selectivity and excellent precision characteristics, with RSD less than 11.9% in the concentrations, in intra- and interassay precision studies. Linearity was proved for a range from 4.00 to 30.00 mg/L and the recovery has been investigated at four different fortification levels, from 89 to 113%. Several approaches found in the literature were used to determinate the LODs and LOQs. Though all strategies used were appropriate, we obtained different values when using different methods. Estimating the S/N ratio with the mean noise level in themigration time of each fluoroquinolones turned out as the best studied method for evaluating the LODs and LOQs, and the values were in a range of 1.55 to 4.55 mg/L and 5.17 to 9.62 mg/L, respectively.