The effects of various levels of chili pepper, Sichuan pepper, and black pepper on the amounts of 17 heterocyclic amines (HAs) from seven categories of both free and protein-bound states in roast beef patties were assessed by ultra-performance liquid chromatography-tandem mass spectrometry combined with principal component analysis. Three groups of HA, including imidazopyridines (DMIP), imidazoquinoxalines (MeIQx and 4,8-MeIQx), and β-carbolines (norharman and harman), were detected and quantified in both their free and protein-bound states, whereas PhIP was detected only in its free state, and imidazoquinolines (IQ, IQ[4,5-b], and MeIQ), α-carbolines (AαC and MeAαC), and phenylpyridines (Phe-P-1) were detected only in their protein-bound states. The results demonstrate that the peppers at all three levels had significant inhibitory effects on free PhIP, DMIP, MeIQx, and 4,8-DiMeIQx and could promote free norharman. Harman was significantly suppressed by chili pepper and black pepper, but enhanced by Sichuan pepper. All 11 protein-bound HAs, with the exception of IQ, IQ[4,5-b], and MeIQx with added chili pepper, were significantly reduced by the three peppers. The total amounts of the free and protein-bound states of all 11 HAs (1692.4 ± 78.9 ng g-1), imidazopyridines (5.5 ± 0.2 ng g-1), imidazoquinolines (7.2 ± 0.2 ng g-1), imidazoquinoxalines (6.9 ± 0.2 ng g-1), α-carbolines (20.1 ± 0.4 ng g-1), and β-carbolines (1651.7 ± 79.5 ng g-1) were suppressed by each level of all of the three peppers except for 0.5% and 1.0% chili pepper. Our findings may facilitate the inhibition of HA formation in the processing of meat products.