The study of phospholipid phase transitions is important for understanding drug- and protein-membrane 14 interactions as well as other phenomena such as trans-membrane diffusion and vesicle fusion. A temperature-controlled stage on a confocal Raman microscope has allowed phase transitions in optically trapped phospholipid vesicles to be monitored. Raman spectra were acquired and analyzed using self-modeling curve resolution, a multivariate statistical analysis technique. This method revealed the subtle spectral changes indicative of sub- and pretransitions and main transitions in vesicles composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The Raman scattering results were compared to differential scanning calorimetry (DSC) experiments and found to be in good agreement. This method of observing lipid phase transition profiles requires little sample preparation and a minimal amount of lipid (<= 0.1 nmol) for an experiment. The conformational changes of the phospholipid molecules occurring during phase transitions are elucidated from the Raman spectroscopy results. The evolution of chain decoupling, rotational disorder, and gauche defects in the lipid acyl chains as a function of temperature is described.