Plant ER membranes are the major site of biosynthesis of several lipid families (phospholipids, sphingolipids, neutral lipids such as sterols and triacylglycerols). The structural diversity of lipids presents considerable challenges to comprehensive lipid analysis. This chapter will briefly review the various biosynthetic pathways and will detail several aspects of the lipid analysis: lipid extraction, handling, separation, detection, identification, and data presentation. The different tools/approaches used for lipid analysis will also be discussed in relation to the studies to be carried out on lipid metabolism and function.