The objective of this study was to determine the use of phytone peptone to optimize the growth and cell density of Lactobacillus reuteri. Four strains of L. reuteri (DSM 20016, SD 2112, CF 2-7F, and MF 2-3,) were used in this study. An overnight culture of individual strains was inoculated into fresh basal media with various protein sources (peptone, tryptone, proteose peptone #3, phytone peptone, tryptic soy broth, yeast extract, and beef extract). Samples were then mixed well and incubated at 37 °C for 15 h. Bacterial growth was monitored by measuring turbidity (optical density 610 nm) at different time intervals during the incubation period. At the end of incubation, samples were plated on de-Man Rogosa Sharpe (MRS) agar to determine the bacterial population. Our results showed that phytone peptone promoted the growth of L. reuteri (p < 0.05) by 1.4 log CFU/mL on average compared to the control samples. Therefore, phytone peptone could be included in laboratory media to enhance growth and increase the cell density of L. reuteri.