Solid-phase microextraction (SPME) coupled to gas chromatography/isotope ratio mass spectrometry (GC/IRMS) was used to elucidate the effects of N-atom protonation on the analysis of N and C isotope signatures of selected aromatic amines. Precise and accurate isotope ratios were measured using polydimethylsiloxane/divinylbenzene (PDMS/DVB) as the SPME fiber material at solution pH-values that exceeded the pK_a of the substituted aniline’s conjugate acid by two pH-units. Deviations of δ¹⁵N and δ¹³C-values from reference measurements by elemental analyzer IRMS were small (<0.9‰) and within the typical uncertainties of isotope ratio measurements by SPME-GC/IRMS. Under these conditions, the detection limits for accurate isotope ratio measurements were between 0.64 and 2.1 mg L⁻¹ for δ¹⁵N and between 0.13 and 0.54 mg L⁻¹ for δ¹³C, respectively. Substantial inverse N isotope fractionation was observed by SPME-GC/IRMS as the fraction of protonated species increased with decreasing pH leading to deviations of −20‰ while the corresponding δ¹³C-values were largely invariant. From isotope ratio analysis at different solution pHs and theoretical calculations by density functional theory, we derived equilibrium isotope effects, EIEs, pertinent to aromatic amine protonation of 0.980 and 1.001 for N and C, respectively, which were very similar for all compounds investigated. Our work shows that N-atom protonation can compromise accurate compound-specific N isotope analysis of aromatic amines.