The present study focused on the development of a new method to determine the lag phase of Lactobacillus in breast milk which was attained during the 1st, 3rd, and 6th month (M1, M3, and M6). The colonies’ phylogenetic analysis, derived from the 16S rRNA gene sequences, was evaluated with genus Lactobacillus pentosus and achieved a similarity value of 99%. Raman spectroscopy in optical densities of 600 nm (OD600) were used for six consecutive days to observe the changes of the cell growth rate. The values of OD600 were well fitted with the regression model. From this work, M1 was found to be the longest lag phase in 18 h, and it was 17% to 27% longer compared to M3 and M6, respectively. However, the samples of M3 and M6 showed the shortest duration in reaching 0.5 of OD600 nm (16 h) which was enhanced by 80% and 96% compared to M1, respectively. These studies will be of significance when applied in determining the bacteria growth curve and in assessing the growth behavior for the strain in human breast milk.