A simple and sensitive liquid chromatography-tandem mass spectrometry (negative ion-electrospray ionization) methodology to determine sphingosine 1-phosphate (S1P) and sphinganine 1-phosphate (DH-S1P) in biological samples is described. The method has been validated over the linearity range of 2-100ng/ml (r>0.999) using synthetic C(17)-sphingosine 1-phosphate (C17-S1P) as an internal standard. In multiple reaction monitoring analysis (378.2>79.2), the lower limit of quantification for S1P was 5.0ng/ml but the detection limit for the bioactive lipid was below 5pg (12fmol). Chromatographic separation was achieved on a UPLC BEH Hilic column with a binary mobile phase consisting of 30mM ammonium acetate (pH 4.0) and acetonitrile/MeOH/30mM ammonium acetate buffer (pH 4.0). The methodology detected 176.7+/-54.0ng/ml of S1P and 81.2+/-23.3ng/ml of DH-S1P in human plasma, as well as 201.0+/-72.0ng/ml of S1P and 96.5+/-20.1ng/ml of DH-S1P in mice plasma.