The aim of the present study was to validate HPLC method for analysis bromelain levels in pineapple water and application that method to determine the storage time of pineapple water. Methods: The reversed phase of HPLC method was tested and optimized before it is validated. The composition and the flow rate of the mobile phase is the optimized parameter. The analytical parameters validated were detection limits, linearity, accuracy and precision. Pineapple water was stored for 8 h at 10ºC and bromelain was determined using the validated HPLC method. Result: The optimum mobile phase composition was methanolwater (70:30) with a flow rate of 1 ml/min. The detector concentration-response was linear with coeffcient of determination of 0.993. The accuracy of HPLC method at a recovery of 1 and 2% bromelain was 106.37 ± 1.94% and 98.12 ± 1.29% (n = 5), respectively. The precision, expressed as the coeffcients of variation (CV), at 1 and 2% bromelain were 1.83 and 1.32% (n = 5), respectively. Bromelain level at zero time was 81.53%. After storage for 8 h at 10ºC, bromelain levels in pineapple juice appeared to decrease not statistically signifcant (p > 0.05), with to mean value of 78.46 ± 2.88%. Conclusion: The HPLC method developed was valid to analyze accurately concentrations of bromelain in pineapple water and it can be used to study the shelf life of pineapple water based on bromelain content. The bromelain content in pineapple juice was not statistically signifcantly different after 8 h storage at 10°C.