Display omitted •MCR-ALS with EFA is applied in the analysis of lipid bilayers FT-IR spectra.•Phase transition temperatures are obtained from various signatures of lipids.•All lipid signatures give equal values of the main phase transition temperatures.•Different lipid parts display slightly different pretransition temperatures.•Possibility for separation of the events related with pretransition is suggested. Temperature-dependent transmission FT-IR spectroscopy and DSC measurements were conducted on lipid multibilayers constituted from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. Lipid multibilayers made from 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, which do not form a ripple phase, were examined as a reference. Spectra were analyzed using multivariate curve resolution technique with alternating least squares and evolving factor analysis (MCR-ALS with EFA) and lipid phase transition temperatures were determined. Polar parts of lipid molecules exert greater response on a ripple phase formation than non-polar ones. However, vibrational signatures of hydrocarbon chains with intramolecular origins display certain qualitative differences that pave the way for future work oriented on uncoupling the events that drive ripple phase formation.