This paper studies the in-column detection performance for chromatography separation. Numerical modeling was performed to investigate the detection efficiency of the electrodes that are suspended within a packed chromatography column. The results show that the density of the silica beads packed within the channel does not hinder the detection ability of the electrode. The findings have been further validated with experimental works. An 8 in-column electrode array provides the full separation progress traces of the five neurotransmitters and metabolites (adrenalin, dopamine, DOPAC, serotonin and 5-HIAA) within the column. From the in-column detection, better peak resolutions are developed though the separation column as compared to the conventional post-column detection. The whole progress of the separation shows that good separation (R s > 1) can be obtained at E5 (28.7 mm from column inlet), whereas baseline separation could be obtained at E8 (last in-column electrode) with R s ≥ 1.5 within 60 min. The presented results suggest that the packing materials within the column do not obstruct the efficiency of the electrodes but able to produce a good baseline separation detection as compared to conventional post-column detection.