Immuno-SERS microscopy (iSERS) is an emerging immuno-based method for protein localization on cells and tissue sections. Antibodies bind specifically to certain proteins that are expressed in the cells or tissues. The antibodies are labeled with plasmonically active noble metal nanoparticles, which are functionalized with Raman-active molecules. The detection is carried out by surface-enhanced Raman scattering (SERS). In this study, gold/gold core/satellite particles (AuKSP) were used as SERS nanotags, which are conjugated to antibodies. The AuKSP consist of positively charged core and negatively charged satellite particles, which were assembled through electrostatic interactions. AuKSP, consisting of 50 nm core and 20 nm satellites, were separated using Asymmetric Flow-Field Flow Fractionation and analyzed online via light absorption and scattering. At first, a separation method for these nanoparticles was developed and optimized. The analysis of the fractograms, which represent the separation behavior of the nanoparticles, exhibit differences in quality between different batches of nanoparticles. The AuKSP were then functionalized with a polyethylene glycol (PEG) shell and conjugated to antibodies in order to localize the predictive biomarker PD-L1 in single-cell experiments. The expression of PD-L1 in cancer tissue strongly correlates with the patients’ response to immunotherapy with checkpoint inhibitors. The localization of this antigen is therefore of great importance. The binding specificity of the SERS-labeled antibodies was additionally confirmed using a negative control experiment. Finally, a proof-of-concept study was carried out, in which multicolor localization of a protein was demonstrated using iSERS microscopy. First, smaller AuKSP were synthesized (30 + 17 nm) and six different Raman reporter molecules were selected. Six spectrally distinguishable SERS nanotags with a similar SERS signal intensity were synthesized. An antibody (anti-HER2) was conjugated to each of the six different SERS nanotags. Subsequently, the prominent biomarker HER2 could be localized on individual SkBr-3 cells using six different SERS nanotag/anti HER2 antibody conjugates. This study is the first step towards the parallel localisation of several proteins on the same cell or tissue section.