A　novel　glutathione　(GSH)-silica　hybrid　monolithic　column　synthesized　via　a　combination　of　thiol-ene　click　reaction　and　one-pot　process　was　described,　where　thiol-end　GSH　organic　monomer　and　2,2-azobisisobutyronitrile　(AIBN)　were　mixed　with　hydrolyzed　tetramethyloxysilane　(TMOS)　and　γ-methacryloxypropyltrimethoxysilane　(γ-MAPS)　and　then　introduced　into　a　fused-silica　capillary　for　simultaneous　polycondensation　and　＂thiol-ene＂　click　reaction　to　form　the　GSH-silica　hybrid　monolith.　The　effects　of　the　molar　ratio　of　TMOS/γ-MAPS,　the　amount　of　GSH,　and　the　volume　of　porogen　on　the　morphology,　permeability　and　pore　properties　of　the　prepared　GSH-silica　hybrid　monoliths　were　studied　in　detail.　A　uniform　monolithic　network　with　high　porosity　was　obtained.　A　series　of　test　compounds　including　alkylbenzenes,　amides,　and　anilines　were　used　to　evaluate　the　retention　behaviors　of　the　GSH-silica　hybrid　monolithic　column.　The　results　demonstrated　that　the　prepared　GSH-silica　hybrid　monolith　exhibited　multiple　interactions　including　hydrophobicity,　hydrophilicity,　as　well　as　cation　exchange　interaction.　The　run-to-run,　column-to-column　and　batch-to-batch　reproducibilities　of　the　GSH-silica　hybrid　monolith　for　phenols＇　retention　were　satisfactory　with　the　relative　standard　deviations　(RSDs)　less　than　1.3%　(n=　5),　2.6%　(n=　3)　and　3.2%　(n=　3),　respectively,　indicating　the　effectiveness　and　practicability　of　the　proposed　method.　In　addition,　the　GSH-silica　hybrid　monolith　was　applied　to　the　separation　of　nucleotides,　peptides　and　protein　tryptic　digests,　respectively.　The　successful　applications　suggested　the　potential　of　the　GSH-silica　hybrid　monolith　in　complex　sample　analysis.　©　2014　Elsevier　B.V.