Liquid chromatography coupled to atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) mass spectrometry (MS), in parallel, was used for detection of bovine brain and chicken egg sphingolipids (SLs). APCI–MS mass spectra exhibited mostly ceramide-like fragment ions, [Cer-H2O + H]+ and [Cer-2H2O + H]+, whereas ESI–MS produced mostly intact protonated molecules, [M + H]+. APCI–MS/MS and MS3 were used to differentiate between isobaric SLs. APCI–MS/MS mass spectra exhibited long-chain base related fragments, [LCB]+ and [LCB-H2O]+, that allowed the sphinganine backbone to be differentiated from the sphingenine backbone. Fragments formed from the fatty amide chain, [FA(long)]+ and [FA(short)]+, allowed an overall fatty acid composition to be determined. The presence of both dihydrosphingomyelin (DSM) and sphingomyelin (SM) sphingolipid classes was confirmed using 31P NMR spectroscopy.