A reliable high-performance liquid chromatography-refractive index detection method for the simultaneous analysis of glucose, methanol, and glycerol in a bioprocess fermentation media using direct injection is presented. The validation studies show a satisfactory selectivity, linearity, accuracy, and recovery of the method. The lowest concentration detectable for glucose and methanol is 3.5 and 6.7 mg/100 mL, respectively. This method could be an attractive choice for the analysis of these compounds not only in fermentation media but also in biomedical and environmental samples.