Tryptophan is one of the most extensively used amino acids in livestock industry owing to its effectiveness in enhancing the growth performance of animals. Conventionally, the production of tryptophan relies heavily on genetically modified Escherichia coli but its pathogenicity is a great concern. Our recent study demonstrated that a lactic acid bacterium (LAB), Pediococcus acidilactici TP-6 that isolated from Malaysian food was a promising tryptophan producer. However, the tryptophan production must enhance further for viable industrial application. Hence, the current study evaluated the effects of medium components and optimized the medium composition for tryptophan production by P. acidilactici TP-6 statistically using Plackett-Burman Design, and Central Composite Design. The optimized medium containing molasses (14.06 g/L), meat extract (23.68 g/L), urea (5.56 g/L) and FeSO4 (0.024 g/L) significantly enhanced the tryptophan production by 150% as compared to the control de Man, Rogosa and Sharpe medium. The findings obtained in this study revealed that rapid evaluation and effective optimization of medium composition governing tryptophan production by P. acidilactici TP-6 were feasible via statistical approaches. Additionally, the current findings reveal the potential of utilizing LAB as a safer alternative tryptophan producer and provides insight for future exploitation of various amino acid productions by LAB.