The　separation　of　chiral　amino　acids　(AAs)　and　their　derivatives　has　always　been　a　research　difficulty　in　the　field　of　biochemistry　due　to　the　high　similarity　of　enantiomeric　structures.　In　this　work,　a　simple　and　quick　method　using　natamycin　(Nat)　as　chiral　selector　has　been　developed　to　simultaneously　separate　chiral　AAs　and　their　derivatives　of　carbobenzoxy/benzyl-AAs　(Cbz/Bzl-AAs)　by　trapped　ion　mobility　spectrometry-mass　spectrometry　(TIMS-MS).　Specifically,　12　groups　of　the　Cbz-AAs　and　Bzl-AAs　can　get　baseline　mobility　separation　by　simple　mixing　with　Nat　to　form　binary　diastereomeric　complex　ions　[Nat+(Cbz-D/L-AA)+H]+　and　[Nat+(Bzl-D/L-AA)+H]+.　While　for　the　remained　5　groups　of　Bzl-D/L-AAs　and　16　groups　of　D/L-AAs　with　unsatisfying　separation,　by　further　adding　P-toluenesulfonic　acid　(PTS),　the　formed　ternary　complexes　can　allow　their　baseline　chiral　separation.　Specifically,　Bzl-D-AAs　and　Bzl-L-AAs　get　much　improved　separation　effect　by　the　formed　diastereomeric　complexes　of　[Nat+(Bzl-AA)2+PTS2+H]+,　which　the　Rp-p　was　improved　from　0　to　2.40;　while　the　D/L-AAs　can　get　baseline　separation　by　the　formed　diastereomeric　complexes　of　[Nat　+　AA　+　PTS　+　H]+,　[Nat　+　AA+(PTS)2+H]+,　and　[Nat+(AA)2+(PTS)2+H]+,　with　the　Rp-p　ranged　from　0.44　to　3.53.　Definitely,　PTS　is　the　first　time　reported　as　the　ligand　to　improve　the　separation　effect　for　the　enantiomers,　and　with　the　higher　assembly　of　chiral　analyte,　Nat,　and　PTS　might　enable　better　chiral　separation　for　the　chiral　amino　acid　and　their　derivatives.　Moreover,　method　validation　of　relative　quantification　and　accuracy　for　the　D/L-AA　and　their　derivatives　were　measured　in　a　mixture,　yielding　R2　greater　than　0.99　and　RSD%　≤　2.68%.　Overall,　Nat　and　PTS　as　chiral　selector　and　ligand　can　be　widely　used　for　chiral　AAs　and　their　derivatives　mobility　separation,　and　potentially　for　the　separation　of　other　AA-related　chiral　molecules.　©　2022　Elsevier　B.V.