The piperamides profile of Piper nigrum cultivated in Costa Rica was studied using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight high-resolution mass spectrometry (UPLC-ESI-QTOF-HRMS) on enriched-piperamides extracts. A total of 31 different piperamides were identified, 24 of them with a methylenedioxyphenyl moiety, including piperine and nine other compounds with the characteristic piperidine ring, as well as guineensine, retrofractamide B, and eight other piperamides with an N-isobutyl group. In addition, piperyline and two other compounds with a pyrrolidine ring, as well as piperflaviflorine B, holding a N-2-methylbutyl chain, were characterized. In turn, pellitorine and six other piperamides exhibiting a long olefinic chain instead of the methylenedioxyphenyl group were also tentatively identified. In addition, quantification was performed using UPLC coupled with a diode array detector (UPLC-DAD), with 15 piperamides being quantified, including piperine, piperyline, piperanine, and piperloguminine with values within the range of previous reports, while results obtained for guineensine (276.5–421.0 mg/100 g dry material) and pellitorine (414.4–725.0 mg/100 g dry material) were higher than those reported in the literature. Additionally, preparative and semi-preparative high-performance liquid chromatography (HPLC) separations allowed to isolate, besides piperine, four other piperamides, which were identified through HRMS, 1H and 13C nuclear magnetic resonance (NMR). These included retrofractamide B, guineensine, pellitorine, and (2E,4E,12Z)-N-isobutyl-octadeca-2,4,12-trienamide, with yields of 134.0 mg/100 g dry material, 209.7 mg/100 g dry material, 361.8 mg/100 g dry material and 467.0 mg/100 g dry material, respectively, with all these values higher than those reported in previous studies in the literature. The findings constitute the first report of such a number and diversity of compounds in P. nigrum cultivated in Costa Rica.