Background: Bromelain is the largest protease enzyme in pineapple (Ananas comosus (L) Merr). For determining the shelf life of pineapple fruit water based on bromelain, the valid method is required. Purpose: The aims of research are to validate high performance liquid chromatography (HPLC) for analysis bromelain levels on pineapple water and application that method to determine the storage time of pineapple water. Methods: The reversed phase of HPLC was optimized before it is validated. The selection of the composition and the flow rate of the mobile phase is the optimized parameter. Meanwhile, validated analytic parameters were linearity, accuracy, and precision. Pineapple water was stored for 8 hours at 10ºC and bromelain was determined using valid HPLC. Result: The optimum mobile phase composition was methanol-water (70:30) with a flow rate of 1ml / min. Response of detector to analytical concentration was linear with coefficient of determination of 0.993. The accuracy of HPLC method based on the recovery of 1 and 2% bromelain were 106.37±1.94% and 98.12±1.29% (n=5), respectively. Precision is expressed as the coefficient of variation (CV). The result showed that the CV values at the addition of 1 and 2% bromelain were 1.83 and 1.32% (n=5). Bromelain levels in pineapple juice showed decrease after storage for 8 hours at 10ᵒC. The result of statistical test showed that the decrease of bromelain content was not significantly different, with mean of (78.46 ± 2.88) %. Conclusion: The developed HPLC method was valid to analysis of bromelain in pineapple water and can be used for the study of shelf life of pineapple water based on bromelain content. The bromelain content in pineapple juice was not differ significantly after 8 hours storage at 10°C.