The metabolites of coconut water stored at room temperature were analyzed using UPLC-MS/MS and multivariate statistical analysis to identify the differential biomarkers and metabolic pathways during post-harvest storage. Principal component analysis (PCA), partial least squares-discriminate analysis (PLS-DA), and orthogonal projections to latent structures discriminant analysis (OPLS-DA) were employed to analyze the UPLC-MS/MS data set of 34 matured coconut water samples collected after 0, 1, 2, 3, 4, and 5 months of storage (MOS); moreover, the p-value and fold change were chosen to identify the differential biomarkers; furthermore, a KEGG pathway was applied to analyze the metabolic pathways. All samples were discriminated well in the OPLS-DA model and were divided into two clusters: groups A (0 MOS, and so on), B, C, and D were in one cluster, and groups E and F were in another. A total of 18 biomarkers were identified among all groups and 12 biomarkers between groups A and E, from which we concluded that the post-harvest storage life of matured coconut water shall not exceed 3 months and the pathways of the TCA cycle, protein hydrolysis from coconut meat, and interconversion among amino acids were mainly enriched during the post-harvest storage.