An analytical method to determine the content of tryptophan (Trp) and tyrosine (Tyr) enantiomers in pumpkin (Cucurbita moschata) is developed and validated. The method consists of a sample preparation procedure with a direct liquid chromatographic separation of enantiomers on a Whelk-O2 chiral stationary phase. The chromatographic separation was performed under gradient reversed-phase conditions in which the effect of pH and organic modifier were studied. A multivariate design of experiments was applied in the optimization of the extraction procedure. After optimization the complete analytical process was validated. Great accuracy was achieved with recovery between 92% and 109% with a relative standard deviation of less than 10%. Limits of detection obtained ranged between 0.04 and 0.07 mg L^-1. The resulting analytical method is applied with success to dose the content of Trp and Tyr and their enantiomers in several pumpkin cultivars. The impact of several traditional cooking methods on the content of these amino acids was also studied. The obtained data showed that studied pumpkin is a good source of free Trp and Tyr, mainly L-Trp. The results confirm the natural presence of D-Trp in this vegetable. Finally, the results demonstrate that the levels of all analytes are modified during traditional cooking treatments.