Abstract: OBJECTIVE To construct a method for simultaneous determination of 12 nucleosides and 18 amino acids in Sepiae Endoconcha. METHODS The analysis was performed on a Acquity BEH Amide column(2.1 mm×100 mm, 1.7 μm) with elution by mobile phase of acetonitrile-0.1% formic acid at a flow rate of 0.3 mL·min^-1 with the column temperature at 30℃ by an ultrafast liquid chromatography coupled with mass spectrometer(UFLC-MS/MS). In addition, the positive ion multiple reaction monitoring mode was used. Principal component analysis and two-dimensional principal component analysis were used to comprehensively evaluate the samples. RESULTS All 12 nucleosides and 18 amino acids showed good linearity (r²>0.999 0), the RSDs of the stability, precision and reproducibility tests were less than 3.24%. The recoveries were in the range of 95.14%-103.84% and RSDs were in the range of 0.93%-3.82%. The total nucleoside content and amino acid content in 18 batches of Sepiae Endoconcha were 82.38-565.65 μg·g^-1 and 1 233.12-9 913.49 μg·g^-1. PCA analysis showed that the main components of Sepiae Endoconcha from different areas contained 9 amino acids and 7 nucleosides, and showed regional characteristics. CONCLUSION This method is accurate, rapid and efficient, and can simultaneously determine 12 nucleosides and 18 amino acids in Sepiae Endoconcha, which can provide relevant basis for quality evaluation and identification of Sepiae Endoconcha, provide reference for subsequent studies.