The determination of active motor threshold (AMT) is a critical step in transcranial magnetic stimulation (TMS) research protocols involving voluntary muscle contractions. As AMT is frequently determined using an absolute electromyographic (EMG) threshold (e.g., 200 microvolts peak-to-peak amplitude), wide variation in EMG recordings across participants has given reason to consider a relative threshold (e.g., = 2x background EMG) for AMT determination. However, these approaches have never been systemically compared. PURPOSE: We sought to compare the AMT values derived from absolute and relative criteria commonly used to determine AMT in the quadriceps muscles, and assess the test-retest reliability of each approach (absolute = 200 microvolts vs. relative = 2x background EMG). METHODS: Eighteen young adults (9 males and 9 females; mean +/- SD age = 23 +/- 2 years) visited the research laboratory on two occasions. All testing was conducted on the dominant limb. During each laboratory visit, maximal voluntary isometric contraction (MVIC) quadriceps torque was measured, with all subsequent TMS procedures conducted as participants maintained 10% of MVIC torque. AMT values were derived from each criterion using motor evoked potentials recorded from the vastus lateralis (VL) and defined as the lowest stimulator output (SO%) needed to meet the specified criteria within at least 5/10 pulses. The order of criteria (i.e., absolute vs. relative) was randomized during the first laboratory visit, and counterbalanced during the second visit. A paired samples t-test, 95% confidence intervals and the effect size were used to compare mean differences in AMT values obtained from each criterion during the second laboratory visit. Paired samples t-tests, intraclass correlation coefficients (ICC2,1), standard errors of measurement (SEMs), and the minimal difference (MD) scores were calculated to assess test-retest reliability of each AMT criterion. RESULTS: Differences between the AMT criteria were small and not statistically significant (absolute criterion mean = 48.9%, relative criterion mean = 47.4%; p = .309, Cohens d = 0.247). The absolute criterion demonstrated moderate to excellent reliability (ICC2,1= .866 [0.648 , 0.950], SEM = 7.9%, MD = 10.4%), but higher AMTs were observed in the second visit compared to the first (p = 0.043). The relative criteria demonstrated good-to-excellent test-retest reliability (ICC2,1= .894 [0.746 , 0.959], SEM = 6.9%, MD = 8.9%) and AMTs were not different between visits (p = 0.420). CONCLUSION: Quantifying AMT with an absolute voltage threshold of 200 microvolts peak-to-peak amplitude and a relative voltage threshold 2x background EMG resulted in similar values within a single testing session. However, the relative voltage criterion demonstrated superior test-rest reliability. TMS researchers aiming to track cortical or corticospinal characteristics across visits should consider implementing relative criterion approaches during their AMT determination protocol.