For the simultaneous detection and determination of the absolute configuration of a thiazole-containing (Tzl-) amino acid in a peptide, we have developed a reliable method using the ‘advanced Marfey's method’, which includes HPLC with a rational guideline, a sensitive derivatizing reagent, 1-fluoro-2,4-dinitrophenyl-5-l-leucinamide (l-FDLA), and a racemization procedure using dl-FDLA for determination of the absolute configuration of constituent amino acids in a peptide. Tzl-amino acids could be directly detected in the hydrolysate by this method, although they were racemized under ordinary hydrolysis conditions. In order to depress the racemization, the flash hydrolysis was introduced. As a result, the flash hydrolysis for 1 h was sufficient to detect each constituent amino acid, and it was possible to identify the original peak. Consequently, the absolute configuration of microcyclamide (1) possessing Tzl-amino acids was determined by the advanced Marfey's method combined with flash hydrolysis. Additionally, this method was successfully applied to the simultaneous detection and determination of the absolute configuration of two other naturally occurring peptides, waiakeamide (2) and goadsporin (3). The established method with the flash hydrolysis had an additional advantage in that labile amino acids, such as tryptophan and methionine sulfoxide, during acid hydrolysis can be detected in the intact form.