BACKGROUND AND AIM: Edible bird's nest (EBN) is the nutrient-rich salivary bioproduct produced by swiftlets in Southeast Asia. Currently, researchers are exploring the therapeutic effects of EBN, such as cell growth promotion, antioxidant content, antiviral effects, bone strengthening, eyes care, and neuroprotection bioactivities. The therapeutic effects of EBN have been studied through different extraction methods but the metabolites profile of the EBN in each extract has not yet been elucidated. This study aimed to profile the water-soluble metabolites of EBN prepared in different extraction methods. Subsequently, an extraction method will be selected as an ideal extraction method for untargeted metabolite profiling on the water-soluble metabolites in EBN. MATERIALS AND METHODS: In this study, water-soluble metabolites of EBN extracted by the four extraction methods were subjected to metabolite profiling through liquid chromatography-mass spectrometry (LC-MS). The extraction methods were acid extraction(ABN), pancreatic extraction (EzBN), eHMG extraction, and spray drying of HMG extraction (pHMG). The metabolite profiles, such as the number of metabolites and their identities in each extraction method, were evaluated through LC-MS analysis. RESULTS: The identity of metabolites present in the four extraction methods is inconsistent. Based on LC-MS analysis, only one and six metabolites were extracted differently through EzBN and ABN, respectively, in the first pre-screening. Through the second LC-MS screening on pHMG and eHMG extraction methods, eHMG was selected as an ideal extraction method due to the highest numbers of water-soluble metabolites with an amount of 193 was detected. Besides, eHMG extraction method was able to extract sialic acid and a high percentage of secondary metabolites. CONCLUSION: This study suggests that eHMG is the ideal extraction method for extracting higher number of water-soluble metabolites from EBN and could be further developed as an extraction method for industry application. In addition, this study also has identified the types of primary and secondary metabolites present in EBN.