A highly sensitive paired ion electrospray ionization mass spectrometry (PIESI-MS) approach was developed for the trace determination of sphingolipids. Apart from their structure role, specific sphingolipids can play a role in cell signaling and as disease markers. With the optimal pairing reagents, detection limits ranged from low femtomole to picomole levels for 14 selected sphingolipids. This improved the detection sensitivity of ESI-MS for many of these analytes up to ~4000 times.