This study aimed to enhance natural gamma aminobutyric acid (GABA) production in yoghurt by the addition of simple sugars and commercial prebiotics without the need for pyridoxal 5'-phosphate (PLP) cofactor. The simple sugars induced more GABA production (42.83-58.56 mg/100 g) compared to the prebiotics (34.19-40.51 mg/100 g), with glucose promoting the most GABA production in yoghurt (58.56 mg/100 g) surpassing the control sample with added PLP (48.01 mg/100 g). The yoghurt prepared with glucose also had the highest probiotic count (9.31 log CFU/g). Simulated gastrointestinal digestion of this GABA-rich yoghurt showed a non-significant reduction in GABA content and probiotic viability, demonstrating the resistance towards a highly acidic environment (pH 1.2). Refrigerated storage up to 28 days improved GABA production (83.65 mg/100 g) compared to fresh GABA-rich yoghurt prepared on day 1. In conclusion, the addition of glucose successfully mitigates the over-use of glutamate and omits the use of PLP for increased production of GABA in yoghurt, offering an economical approach to produce a probiotic-rich dairy food with potential anti-hypertensive effects.