A simple procedure is proposed for the determination of the antibiotic moxifloxacin in urine using nanostructured gold as surface-enhanced Raman scattering signal enhancer. The standard addition method in conjunction to multivariate curve resolution-alternating least squares was applied to eliminate the matrix effect and to isolate the spectral contribution of the analyte. Even in the presence of unexpected interferences in the urinary media, it was possible to extract and quantify the analyte response, reaching, in this way, the so-called second-order advantage from first-order data. Moreover, although a saturation phenomenon of the metallic surface was observed, the results of the proposed methodology presented important advantages such as high sensitivity and simpler experimental procedures. The moxifloxacin was determined at levels of 0.70 and 1.50 μg mL(-1) in urine diluted to 1.0% (corresponding to 70.0 and 150 μg mL(-1) in the original samples) with relative errors of 4.23 and 8.70%, respectively. The limit of detection (0.085 μg mL(-1)) and limit of quantification (0.26 μg mL(-1)) values indicated that the quantification can be accomplished in urine up to 24 h after the administration of a single 400-mg dose.