Glycerophospholipids are important structural lipids in membranes with changes associated with progressive neurodegenerative disorders such as Alzheimer disease. Synthesis of the platelet activating factor (PAF) glycerophospholipid subclass is implicated in the control of neuronal differentiation and death. In this article, we combine nanoflow HPLC and mass spectrometry to screen, identify, and quantitate changes in glycerophospholipid subspecies, specifically PAF family members, over the course of neuronal differentiation. Furthermore, precursor ion scans for fragments characteristic of PAF phosphocholine family members and the standard additions of PAF subspecies were combined to perform absolute quantitation of PAF lipids in undifferentiated and differentiated PC12 cells. Surprisingly, a marked asymmetry was detected in the two predominant PAF species (C16:0, C18:0) over the course of differentiation. These results describe a new technique for the sensitive analysis of lipids combining nanoflow HPLC, ESI-MS, and precursor ion scan. Limits of detection of as little as 2 pg of PAF and LPC were obtained, and analysis of the lipidome of as little as 70,000 cells was performed on this system. Furthermore, application to the PC12 model identified a quantifiable difference between PAF molecular species produced over the course of neuronal differentiation.