Abstract: Naturally-occurring precipitate of traditional Chinese medicine (TCM) is generally considered to be a common phenomenon. The formation of naturally-occurring precipitate originates from the interactions among different chemical components during the decoction process. Previous literature has reported the existence of naturally-occurring precipitate in Sini decoction. In this study, seven pairs of acid-base non-covalent complexes in Sini decoction were characterized using cold spray ionization-mass spectrometry (CSI-MS). The CSI device was built in house, where a stream of nitrogen gas was cooled by liquid nitrogen and used as the sheath gas, providing low-temperature ionization conditions for the MS analysis of the acid-base non-covalent complexes. The gas pipeline was equipped with a valve to regulate the nitrogen flow rate. Data acquisition was carried out using a quadrupole/Orbitrap high-resolution mass spectrometer operated in both full-scan MS and collision-induced dissociation MS/MS modes. Experimental results showed that the seven pairs of acid-base non-covalent complexes were unequivocally observed in positive and negative ionization modes. Mass spectrometric responses for each acid-base non-covalent complex varied with different chemical structures, binding abilities and ionization efficiencies. A representative pair of acid-base non-covalent complex (enoxolone/benzoylmesaconine) was selected to investigate the influence of the acid-base concentration ratio (3∶1, 2∶1, 3∶2, 1∶1, 2∶3, 1∶2, 1∶3) on the binding stoichiometry. The enoxolone/benzoylmesaconine complexes with the binding ratios of 1∶1, 1∶2, 2∶1 were observed, among which the mass spectrometric peak of the 1∶1 binding ratio was the most intensive. The non-covalent interactions of the representative enoxolone/benzoylmesaconine complex were analyzed using isothermal titration calorimetry (ITC) and computational chemistry. The thermodynamic parameters were obtained in the ITC experiment. In comparison with entropy change, the contribution of enthalpy change to Gibbs free energy was more significant, indicating that Van der Waals force and hydrogen bonding might be the main driving force in the formation of acid-base non-covalent complexes. This result was further confirmed by the docking model and simulation results of computational chemistry. Multiple hydrogen bonds derived from the interactions between specific atoms locating in the acid and base compounds promoted the self-assembly process. Moreover, Van der Waals attraction or repulsion force was generated between the C and H atoms at different positions, which in conjunction with hydrogen bonding interaction drove the formation of the acid-base non-covalent complexes. This study provides theoretical foundation and technical instruction for exploring the formation mechanism of naturally-occurring precipitate in Sini decoction.