Peptide therapeutics have grown popular over the past decade because of their wide applications in medicine. One of the problems of peptide therapeutic is the low solubility in water and organic solvents. Poorly soluble peptides have many problems associated to analytical characterization, purification, and applications. In this research, chromatographic separation of the hydrophobic Temporin-L peptide in menthol based deep eutectic solvent (DES) is investigated. Deep eutectic solvent (DES) is the alternative greener solvent and has similar characteristics of ionic liquids, but they are cheaper to synthesis, less toxic and environmentally friendly, recyclable, biodegradable, and are suitable for the biological system. Temporin-L (FVQWFSKFLGRIL) is known to be a very hydrophobic peptide, due to the presence of leucine, isoleucine, and phenylalanine. To assess the impact of menthol-thymol DES on the Temporin L, various samples were prepared by adding 1%, 5%, 10%, and 20% of DES. The Agilent 1290 infinity II UHPLC system equipped with a binary pump, a multisampler and a UV detector excitation at 230 nm was used for all experiments. ZORBAX C18 column (2.1x50 mm, 1.8 Micron) was utilized. Mobile phase consisted of water (A) and acetonitrile (B) with 0.01% TFA. The flow rate was set at 1 mL/min. In menthol-acetic acid- DES, the retention time, peak area, and height of the peptides did not significantly change when DES amount was increased. However, significant changes are noticed for menthol-thymol DES. The peak area and height of the peptide are drastically increased to 11,198 (mAU路s) and 2001 (mAU), respectively when the peptide was dissolved in 1% menthol-thymol DES compared to water (peak area: 537 mAU路s and height: 132 mAU). Moreover, similar trend was also observed when peptide was dissolved in 5%-20% DES. This study discloses that menthol-thymol DES can deliver the best chromatographic separation, recovery, and resolution for the hydrophobic peptides