A cation-selective microfluidic sample preconcentration system is described. The cation sample was electropreconcentrated using a reversed-direction electroosmotic flow (EOF) and an anion-permselective filter, where an electric double layer (EDL) overlap condition existed. The anion-permselective filter between microchannels was fabricated by three different methods: 1) extending a positively charged, nanoporous, polymer membrane by photopolymerization of poly(diallyldimethylammonium chloride) (PDADMAC); 2) etching a nanochannel and then coating it with a positively-charged monomer, N-[3-(trimethoxysilyl)propyl]-N′-(4-vinylbenzyl)ethylenediamine hydrochloride (TMSVE); and, 3) etching a nanochannel and then coating it with a positively-charged, pre-formed polymer, polyE-323. The EOF direction in the microchannel was reversed by both TMSVE and polyE-323 coatings. The cation-selective preconcentration was investigated using charged fluorescent dyes and tetramethylrhodamine isothiocyanate (TRITC)-tagged peptides/proteins. The preconcentration in the three different systems was compared with respect to efficiency, dependence on buffer concentration and pH, tolerable flow rate, and sample adsorption. Both TMSVE- and polyE-323-coated nanochannels showed robust preconcentration at high flow rates, whereas the PDADMAC membrane maintained anion-permselectivity at higher buffer concentrations. The TMSVE-coated nanochannels showed a more stable preconcentration process, whereas the polyE-323-coated nanochannels showed a lower peptide sample adsorption and robust efficiency under a wide range of buffer pHs. The system described here can potentially be used for the preconcentration of cationic peptides/proteins on microfluidic devices for subsequent analyses.