Our goal was to determine the minor components in total capsaicinoids isolated from red chili pepper Capsicum annuum L. growing in the Fergan Valley, Kokand, Uzbekistan. Total capsaicinoids were isolated using petroleum ether extraction followed by column chromatography over a C18 hydrophobic silochrom sorbent, with a yield of 0.65 ± 0.05% per dry weight of the starting material. Pure capsaicinoids were separated by HPLC on a Zorbax 300SB-C18 column (4.6 × 250 mm) using a mobile phase of 0.1% trifluoroacetic acid (TFA) and acetonitrile (CH3CN) with gradient elution (5% CH3CN for 0–5 min, 5–60% CH3CN for 5–35 min, 60% CH3CN for 35–40 min, and a reverse gradient to 5% CH3CN over 5 min) at a flow rate of 1 mL/min and detection at 269 nm. Capsaicinoids were identified via liquid chromatography-mass spectrometry (LC-MS) using a Waters ZQ-4000 instrument with electrospray ionization (ESI+) under conditions including a drying gas flow rate of 450 L/h, drying gas temperature of 250°C, skimmer cone potentials of 35 and 50 V, and a mass range of 100–400 m/z. Eleven pure compounds were analyzed, with their mass spectra showing characteristic positive ions (e.g., 3-methoxy-4-hydroxybenzylidene at m/z 136.6, 100%). The qualitative and quantitative compositions of these compounds, including their trivial names, radicals, contents (ranging from 45.29% for capsaicin to 0.140% for nordihydrocapsaicin II), and elution times, are presented in Table 1. This study is the first to report the qualitative and quantitative composition of the minor constituents of total capsaicinoids from Capsicum annuum grown in Uzbekistan.