Sphingolipids, a major component of hair lipids can determine the physicochemical properties of human hairs such as the chemical diffusion barrier, water retention and cell cohesion. In this study, we have developed a quantitation method for sphingolipids in animal and human hair. The lipid contents of sphingoid bases, sphingoid bases 1-phosphate, Ceramide (Cer) and dihydroceramide (DHCer) in hair were measured by liquid chromatography linked tandem mass spectrometry (LC-MS/MS). The established method was evaluated against quality parameters such as specificity, carryover, linearity and calibration, correlation of determination (R2), LOD, LOQ, precision, accuracy, and recovery. Specifically, analyzed DHCer levels were higher in normal hair roots to distal tip ends than Cer contents ranged from 1960.23 to 1373.24 pmol/g. Interestingly, DHCer were decreased significantly in alopecia patient upper hair fiber compared to alopecia patient side hair fiber. Specific ceramides (C16:0 and C24:1) in alopecia patient upper hair were observed in very low levels, while Cer was not change in alopecia hair. In experimental mouse model of alopecia hair and skin, Cer and DHCer were significantly low as well. These results suggested that hair DHCer content may be used as a potential biomarker for the progress in alopecia pathogenesis.