Glycolic acid and allantoin are known to be responsible, at least in part, for the main activities of snail slime, such as moisturizing, skin regeneration, antioxidant, soothing and anti-inflammatory. Accordingly, a hydrophilic interaction liquid chromatography (HILIC) method for the analysis of allantoin and glycolic acid in snail slime samples was developed by a conventional UV-Vis detector and the diol-type Luna® HILIC column. An optimized eluent made up of acetonitrile/water/methanol/formic acid (90:5:5:0.1 v/v/v/v) allowed the separation of the two investigated compounds from each other and from lactic acid and hydantoin used as internal standards. Being allantoin a chiral compound, an enantioselective HPLC protocol was also developed. The chiral analysis of allantoin was performed with the Lux® 3 μm i-Amilose-3 chiral stationary phase using the same mobile phase as for the achiral analysis. The chiral method was efficiently transferred to a HPLC system coupled to triple-quadrupole mass spectrometry (MS/MS). HPLC-MS/MS analysis revealed the racemic nature of allantoin in the pure snail slime. The same profile was also observed in a prototype hydrogel prepared with snail slime. Moreover, the same method allowed to measure the amount of glycolic acid in the two investigated samples (pure snail slime and gel) and ascertain a valuable extraction recovery. The enantiomeric elution order (S)<
(R) with the amylose-based phase was established coupling the results of experimental electronic circular dichroism analysis to time-dependent density functional theory simulations.