The signatures of natural stable nitrogen isotopic composition (delta N-15) of individual amino acid (AA) have been confirmed to be a potentially effective tool for elucidating nitrogen cycling and trophic position of various organisms in food webs. In the present study, a two-stage derivatisation approach of esterification followed by acylation was evaluated. The biological samples underwent acid hydrolysis and the released individual AA was derivatived into corresponding N-pivaloyl-isopropyl (NPIP) esters for nitrogen isotopic analysis in gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Usually, 13 individual AA derivatives were separated with fine baseline resolution based on a nonpolar gas chromatography column (DB-5 ms). The minimum sample amount required under the presented conditions is larger than 20 ngN on column in order to accurately determine the delta N-15 values. The delta N-15 values determined by GC-C-IRMS with a precision of better than 1 parts per thousand, were within 1 parts per thousand after empirical correction compared to the corresponding measured by element analysis (EA)-IRMS. Bland-Altman plot showed highly consistency of the delta N-15 values determined by the two measurement techniques. Cation exchange chromatography was applied to remove interfering fraction from the extracts of plant and animal samples and without nitrogen isotope fractionation during the treatment procedure. Moreover, this approach was carried out to estimate the trophic level of various natural organisms in a natural lake environment. Results highly proved that the trophic level estimated via the presented AA method well reflected the actual food web structure in natural environments.