Abstract Background Kidney diseases are a global health problem. Currently, over 2 million people require dialysis or transplant which are associated with high morbidity and mortality; therefore, new researches focused on regenerative medicine have been developed, including the use of stem cells. Results In this research, we generate differentiated kidney cells (DKCs) from mouse pluripotent stem cells (mPSCs) analyzing their morphological, genetic, phenotypic, and spectroscopic characteristics along differentiation, highlighting that there are no reports of the use of Fourier transform infrared (FTIR) spectroscopy to characterize the directed differentiation of mPSCs to DKCs. The genetic and protein experiments proved the obtention of DKCs that passed through the chronological stages of embryonic kidney development. Regarding vibrational spectroscopy analysis by FTIR, bands related with biomolecules were shown on mPSCs and DKCs spectra, observing distinct differences between cell lineages and maturation stages. The second derivative of DKCs spectra showed changes in the protein bands compared to mPSCs. Finally, the principal components analysis obtained from FTIR spectra allowed to characterize chemical and structurally mPSCs and their differentiation process to DKCs in a rapid and non-invasive way. Conclusion Our results indicated that we obtained DKCs from mPSCs, which passed through the chronological stages of embryonic kidney development. Moreover, FTIR spectroscopy resulted in a non-invasive, rapid and precise technic that together with principal component analysis allows to characterize chemical and structurally both kind of cells and also discriminate and determine different stages along the cell differentiation process.