Ananas comosus L. Merr. is asexually propagated through the conventional method from its crowns, slips, hapas, and suckers. Plant tissue culture technique is applied for mass production of clonal pineapple within a shorter period compared to conventional propagation. Hence, this research aims to study the effect of coconut water and optimum sucrose concentration for in vitro growth performance of pineapple. In addition, this research also aims at optimizing the fertilization mechanism for the acclimatization of clonal pineapple ex vitro. The in vitro regeneration was performed by adding a different volume of coconut water and concentration of sucrose into the prepared culture media supplemented with 2.00 mg/L 6-Benzylaminopurine (BAP) and 1.00 mg/L Naphthaleneacetic acid (NAA). For acclimatization method, two different types of fertilizer served as treatments which were compound fertilizers (NPK 15:15:15) and controlled-release fertilizer (AJIB CRF, NPKMg 10:5:18:3). The planting media consisted of topsoil, sand, and compost composition in a ratio of 2:1:2. Results showed that culture media contained 20.00 mL/L coconut water obtained the highest number of shoots (45.33±7.56). On the other hand, culture media supplemented with 30.00 g/L sucrose concentration showed the highest number of shoots (47.25±10.81). Meanwhile, the acclimatization method found that controlled-release fertilizer (AJIB CRF), NPKMg (10:5:18:3) showed about 90% of survival rate. The mean value for the number of leaves and leaves length (cm) in acclimatized clonal pineapple using Controlled-release fertilizer (AJIB CRF) were 5.48±0.96 and 6.00±1.04, respectively. Therefore, the addition of coconut water, optimum sucrose concentration, and the suitable type of fertilizer in both in vitro and ex vitro promote a better growth performance of clonal pineapple.