Surface Enhanced Raman Scattering (SERS) spectroscopy is a rapid and innovative analysis technique involving metallic nanoparticles (NPs). The interaction between NPs and norepinephrine gives an exaltation of the Raman signal under certain experimental conditions. The control of the signal exaltation, crucial for sensitive analyses, remains one of the main limitations of this technique. The aim of this work is to optimize the exaltation conditions for an optimal SERS signal at two concentrations of norepinephrine (NOR) and spherical gold NPs in suspension. This first work will fix the optimal experimental conditions essential for the development of robust discriminant and quantitative analysis of catecholamine. Two complete 3-factors 3-levels experiment designs were performed at 20 µg.mL−1 and 100 µg.mL−1 norepinephrine concentrations, each experiment being repeated 3 times. The optimization factors were the process of synthesis (variation of the quantity of gold and citrate used for the three synthesis SA, SB and SC) and HCl (0.3 M, 0.5 M, 0.7 M) as well as the volume ratio of NPs and norepinephrine (0.5, 2, 3.5) for SERS acquisition. Spectral acquisitions were performed with a handheld Raman spectrometer with an excitation source at 785 nm. For each sample, 31 acquisitions were realized during 3 s every 8 s. The optimization parameter was the intensity of the characteristic band of norepinephrine at 1280 cm−1. A total of 5,042 spectra were acquired and the pre-treatment selected for all spectra was asymmetric least square combined to a smoothing of Savistsky Golay (ALS – SG). The optimal contact time between norepinephrine and NPs depends on the experimental conditions and was determined for each experiment according to the mean intensity between the three replicates. After interpretation of the experimental designs, the optimal conditions retained were the quantity of gold corresponding to SA and the HCl concentration 0.7 M for the two concentrations of norepinephrine. Indeed, the optimal volume ratio depend on the NOR concentration.