Abstract Introduction The P300 test requires well-defined and unique criteria, in addition to training for the examiners, for a uniform analysis of studies and to avoid variations and errors in the interpretation of measurement results. Objectives The objective of this study is to verify whether there are differences in P300 with and without subtraction of traces of standard and nonstandard stimuli. Method We conducted this study in collaboration with two research electrophysiology laboratories. From Laboratory 1, we selected 40 tests of subjects between 7 44 years, from Laboratory 2, we selected 83 tests of subjects between 18 44 years. We first performed the identification with the nonstandard stimuli; then, we subtracted the nonstandard stimuli fromthe standard stimuli. The examiners identified the waves, performing a descriptive and comparative analysis of traces with and without subtraction. Results After a comparative analysis of the traces with and without subtraction, there was no significant difference when compared with analysis of traces in both laboratories, within the conditions, of right ears (p = 0.13 and 0.28 for differences between latency and amplitude measurements) and left ears (p = 0.15 and 0.09 for differences between latency and amplitude measurements) from Laboratory 1. As for Laboratory 2, when investigating both ears, results did not identify significant differences (p = 0.098 and 0.28 for differences between latency and amplitude measurements). Conclusion There was no difference verified in traces with and without subtraction. We suggest the identification of this potential performed through nonstandard stimuli.