Nowadays a quick and inexpensive method, which allows rapid, in vivo comprehensive probiotic bacteria identification, is needed. To elucidate a new concept to evaluate probiotic bacteria, near infrared spectroscopy with aquaphotomics were applied to monitor the growth of eight Lactobacillus bulgaricus and one Lactobacillus gasseri bacteria strains. Their resistance to low pH (1.8) in the presence of pepsin and bile were measured and further used as reference data for analysis of the simultaneously acquired spectral data. The acquired spectral data in the region of 1100–1300 nm were subjected to various methods for multivariate data analyses—principal component analysis, linear discriminant analysis, soft independent modeling of class analogy, and partial least squares regression. The results showed high accuracy of bacteria strains classification according to their resistance and the potential of the tested wavelength region for rapid selection and prediction of some basic phenotypic characteristics of probiotic candidates. Results of the current study also revealed different suitability of each growth stage when using near infrared spectra for the classification of bacteria strains.