This paper reported the optimization of glucose production from ammonia pre-treated oil palm trunk biomass via enzymatic saccharification process using response surface methodology (RSM). A set of experiment was computed by face centered central composite design (FCCCD) using Design Expert software to statistically evaluate the findings. Three independent variables namely: temperature (35 -55 °C), pH (4.5 -6.5) and enzyme ratio (cellullase: β-glucosidase; 3:1 -7:1) were investigated under the given conditions designed by RSM. The experimental result (4.964 ± 0.006 g.L -1 ) was in good agreement with RSM model prediction (4.958 g.L -1 ) with an error less than 0.2 %. The RSM design has been proven to successfully predict the glucose response in this study.Keywords: oil palm trunk biomass, enzymatic saccharification, lignocellulosic substrate, ammonia pre-treament, response surface methodology Abstrak Kertas kerja ini melaporkan pengoptimuman hasilan glukosa daripada prarawatan ammonia biomas batang kelapa sawit melalui proses sakarifikasi enzim dengan menggunakan kaedah gerak balas permukaan (RSM). Satu set eksperimen telah dibentuk iaitu kaedah muka berpusat reka bentuk komposit dengan menggunakan perisian Design Expert bagi menilai penemuan kajian secara statistik. Tiga pembolehubah bebas iaitu: suhu (35 -55 ° C), pH (4.5 -6.5) dan nisbah enzim (selulase: β-glukosidase; 3:1 -7:1) telah disiasat di bawah situasi yang direka oleh RSM. Hasil eksperimen (4.964 ± 0.006 g.L -1 ) yang diperoleh berada dalam persetujuan yang baik dengan ramalan model RSM (4.958 g.L -1 ) iaitu pada ralat kurang daripada 0.2 %. Reka bentuk RSM telah terbukti berjaya meramalkan respon glukosa dalam kajian ini.