Matrix-assisted　laser　desorption/ionization　time-of-flight　mass　spectrometry　(MALDI-TOF　MS)　is　an　excellent　analytical　technique　for　rapid　and　sensitive　analysis　of　macromolecules　such　as　polymers　and　proteins.　However,　the　main　drawback　of　MALDI-TOF　MS　is　its　difficulty　to　detect　small　molecules　with　mass　below　700　Da　because　of　the　intensive　interference　from　MALDI　matrix　in　the　low　mass　region.　In　recent　years　there　has　been　considerable　interest　in　developing　matrix-free　laser　desorption/ionization　by　using　nanostructured　substrates　to　substitute　the　conventional　organic　matrices,　which　is　often　referred　as　surface-assisted　laser　desorption/ionization　time-of-flight　mass　spectrometry　(SALDI-TOF　MS).　Despite　these　attractive　features,　most　of　the　current　SALDI-TOF　MS　for　the　analysis　of　small　molecules　employ　positive　ion　mode,　which　is　subjected　to　produce　multiple　alkali　metal　adducts,　and　thus　increases　the　complexity　of　the　analysis.　Different　from　the　complicated　adducts　produced　in　positive　ion　mode,　mass　spectra　obtained　in　negative　ion　mode　are　featured　by　deprotonated　ion　peaks　without　matrix　interference,　which　simplifies　the　interpretation　of　mass　spectra　and　detection　of　unknown.　In　this　review,　we　critically　survey　recent　advances　in　nanostructured　substrates　for　negative　ion　LDI-TOF　MS　analysis　of　small　molecules　in　the　last　5　years.　Special　emphasis　is　placed　on　the　preparation　of　the　nanostructured　substrates　and　the　results　achieved　in　negative　ion　SALDI-MS.　In　addition,　a　variety　of　promising　applications　including　environmental,　biological,　and　clinical　analysis　are　introduced.　The　ionization　mechanism　of　negative　ionization　is　briefly　discussed.