Abstract: To address the challenges of water-soluble natural flavors, such as complex components, low volatility and limitations of conventional analytical methods, a direct injection analytical method was developed by combining Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) with an atmospheric pressure chemical ionization (APCI) source. Sample pretreatment conditions (sample weight, dilution solvent, etc.) and mass spectrometry parameters (number of scans, flight time, etc.) were optimized to realize rapid acquisition of APCI fingerprint spectra and quality evaluation of water-soluble natural tobacco flavors. This proposed evaluation method was compared with conventional analytical techniques and existing algorithms, the Principal component analysis (PCA) was performed on water-soluble natural flavors of different varieties based on the differences, and five data preprocessing approaches including raw data, maximum bucket value method, and total bucket value method were compared. The results showed that: 1) This method showed excellent precision, repeatability and stability with no chromatographic column separation, high analysis speed (tens of millisecond), a broad mass range (54~1,600 Da) and high mass accuracy. 2) Compared with the existing evaluation methods, the proposed method could effectively eliminate overestimation of evaluation results caused by high response peaks and overcome the deficiency of insensitivity to differential signals of low response peaks, which extremely improved the accuracy of evaluation results. It can provide a rapid and efficient evaluation tool for the quality control of water-soluble natural tobacco flavors for with different batches, sources and varieties during procurement. 3) Differential analysis of water-soluble natural flavors enabled the identification of characteristic ions (exact masses) that contributed greatly to sample differences, and proved core references for subsequent accurate quality control analysis, traceability identification and characteristic component characterization of flavors.