The　differentiation　of　3-AminoPyrrolidine　(3AP)　and　its　derivative　isomers　is　significant　due　to　their　use　as　intermediates　in　drug　synthesis.　However,　the　separation　of　these　isomers,　which　exhibit　both　chiral　and　positional　isomerism,　has　consistently　posed　challenges.　In　this　study,　an　efficient　analytical　method　was　developed　to　discrimination　between　the　four　isomers　of　R/S-I/N-Boc-3-AminoPyrrolidine　(R/S-l/N-Boc-3AP)　and　four　groups　of　derived　chiral　isomers,　namely　R/S-N-Methyl-3-AminoPyrrolidine　(R/S-N-Me-3AP),　R/S-N-Ethyl-3AminoPyrrolidine　(R/S-N-Et-3AP),　R/S-1-Bzl-3-AminoPyrrolidine　(R/S-1-Bzl-3AP),　and　R/S-1-Cbz-3AminoPyrrolidine　(R/S-1-Cbz-3AP).　The　discrimination　was　achieved　by　utilizing　natamycin　(Nat)　as　a　differentiation　agent　and　2/3/4-aminobenzenesulphonic　acid　(2/3/4-ABSA)　as　ligand.　The　facile　discrimination　of　the　four　positional　isomers　of　R/S-1-Boc-3AP　and　R/S-N-Boc-3AP　was　achieved　using　ion　mobility　spectrometry　(IMS)　based　on　their　formed　diastereomeric　complexes　[Nat　+　(Boc-3AP)2　+　(2-ABSA)2　+　H]+.　The　isomers　were　also　studied　via　tandem　mass　spectrometry　(MS/MS)　using　the　formed　ternary　complexes,　different　fragment　ions　were　observed　for　l-Boc-3AP　and　N-Boc-3AP,　but　the　R/S　enantiomers　cannot　be　discriminated　either.　In　addition,　the　enantiomers　R/S-l-Bzl-3AP　and　R/S-l-Cbz-3AP　were　differentiated　based　on　mobility　using　[Nat　+　(R/Sl-Bzl-3AP)　+　(3-ABSA)2　+　H]+　and　[Nat　+　(R/S-l-Cbz-3AP)　+　2-ABSA　+　H]+;　however　for　the　small　enantiomeric　molecules　of　R/S-N-Me-3AP　and　R/S-N-Et-3AP,　chiral　discrimination　was　not　achieved　via　the　ternary　complexes　of　Nat　+　R/S-N-Me-3AP-ABSA　and　Nat　+　R/S-N-Et-3AP-ABSA.　Importantly,　the　separation　was　greatly　improved　by　the　coordinated　alkali　metal　ions,　where　the　separation　resolution　(Rp-p)　increased　with　the　radius　of　the　metal　ions.　Finally,　quantitative　analysis　and　method　validation　were　carried　out　for　the　four　Boc-3AP　isomers　using　both　MS/MS　and　IMS-MS.　Overall,　the　proposed　method　opens　possibilities　for　the　rapid　discrimination　and　quantification　of　isomers　exhibiting　both　chiral　and　positional　isomerism　in　MS/MS　and　IMS-MS　analyses.