Background: Isoniazid (INH) is one of the anti-tuberculosis drugs. The main pathway of INH metabolism is by acetylation and producing acetyl isoniazid (ACINH). Acetylation rate can differ depending on N-acetyltransferase-2 (NAT-2) activity. In addition, INH also undergoes hydrolysis in the liver through enzymatic reactions with cytochrome 2E1 (CYP2E1) and results in the formation of hepatotoxic compounds. Pineapple (Ananas comosus) juice is an inhibitor of CYP2E1. Objective: The purpose of this research was to study the interaction of pineapple juice with the acetylation rate of INH in healthy subjects. Method: Acetylation rate of INH was determined based on the ratio of AcINH and INH levels in human plasma. AcINH and INH levels in the subject plasma were analyzed by high performance liquid chromatography (HPLC). Results: The HPLC system was using the C18 stationary phase column (dimensions of 4.6x250mm), 20 mM hexansulfonic acidic-methanol (65:35) as a mobile phase with a flow rate of 1mL/minute, UV detector at 265nm. The linearity, accuracy, precision, and selectivity of the method fulfills the validation requirements of the bio-analysis method. Healthy subjects (41 of Indonesian) were voluntarily involved in this study. Based on the Wilcoxon test for two related samples statistical analysis at a 95% confidence level, pineapple juice is known to affect the acetylation rate of INH. Pineapple juice can increase the acetylation rate of INH on a slow acetylator and conversely reduce the acetylation rate of INH on a fast acetylator. Conclusion: The influence of pineapple juice to the acetylation rate of INH can be the basis for the development of pineapple juice as an adjuvant to reduce the hepatotoxic effect of INH. The use of pineapple juice can be further investigated in experimental animals before tuberculosis patients.