To determine the origin of N-15-labeled phenylalanine in microbial metabolic flux analysis using N-15 as a tracer, a method for measuring phenylalanine delta N-15 using HPLC coupled with elemental analysis-isotope ratio mass spectrometry (EA-IRMS) was developed. The original source of the N-15-labeled phenylalanine was determined using this new method that consists of three steps: optimization of the HPLC conditions, evaluation of the isotope fractionation effects, and evaluation of the effect of pre-processing on the phenylalanine nitrogen stable isotope. In addition, the use of a N-15-labeled inorganic nitrogen source, rather than N-15-labeled amino acids, was explored using this method. The method described here can also be applied to the analysis of metabolic flux.