A simple, efficient, cost-effective and less time consuming analytical method was developed to separate the enantiomers of certain DL-amino acids using Levofloxacin as chiral selector. Levofloxacin was used as chiral ligand exchange reagent on TLC plates. Chiral separation was achieved under different conditions like pH of reaction mixture, mobile phase, polarities of solution or reaction mixture and temperature. Separation mechanism of the enantioseparation was also discussed.