Compound-specific isotope analysis of nitrogen in amino acids (CSIA-AA, δ¹⁵N_AA) has gained increasing popularity for elucidating energy flow within food chains and determining the trophic positions of various organisms. However, there is a lack of research on the impact of hydrolysis conditions, such as HCl concentration and hydrolysis time, on δ¹⁵N_AA analysis in biota samples. In this study, we investigated two HCl concentrations (6 M and 12 M) and four hydrolysis times (2 h, 6 h, 12 h, and 24 h) for hydrolyzing and derivatizing AAs in reference materials (Tuna) and biological samples of little egret (n = 4), night heron (n = 4), sharpbelly (n = 4) and Algae (n = 1) using the n-pivaloyl-iso-propyl (NPIP) ester approach. A Dowex cation exchange resin was used to purify amino acids before derivatization. We then determined δ¹⁵N_AA values using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). The results revealed no significant differences (p > 0.05) in δ¹⁵N_AA values among samples treated with different HCl concentrations or hydrolysis times, particularly for δ¹⁵N_Glx (range: 21.0–23.5‰) and δ¹⁵N_Phe (range: 4.3–5.4‰) in Tuna (12 M). Trophic positions (TPs) calculated based on δ¹⁵N_AA at 2 h (little egret: 2.9 ± 0.1, night heron: 2.8 ± 0.1, sharpbelly: 2.0 ± 0.1 and Algae: 1.3 ± 0.2) were consistent with those at 24 h (3.1 ± 0.1, 2.8 ± 0.1, 2.2 ± 0.1 and 1.1 ± 0.1, respectively), suggesting that a 2-h hydrolysis time and a 6 M HCl concentration are efficient pretreatment conditions for determining δ¹⁵N_AA and estimating TP. Compared to the currently used hydrolysis conditions (24 h, 6 M), the proposed conditions (2 h, 6 M) accelerated the δ¹⁵N_AA assay, making it faster, more convenient, and more efficient. Further research is needed to simplify the operational processes and reduce the time costs, enabling more efficient applications of CSIA-AA.