Sorbic acid (SA: CH₃–CH=CH–CH=CH–COOH) and its salts are widely used as preservatives in foodstuff because of their growth inhibitory effects on mold, yeast and a wide range of bacteria. However, it is still unclear whether SA and its salts are actually incorporated in these organisms and a higher organisms like mammalian cells. Acidic compounds such as SA are usually analyzed by HPLC with eluents containing acetic acid, formic acid and their ammonium acetates, but such acidic buffers may suppress the ionization efficiency of the acidic compounds in negative-mode electrospray ionization (ESI). In this study, we present a sensitive and simple method for analysis of SA by HPLC with non-acidic solvents such as CH₃CN/CH₃OH–H₂O by negative ion mode ESI-LC/MS. As a result, SA at less as 30 fmol was selectively determined by the selected reaction monitoring (SRM) mode. It was defined as the peak area with a signal-to-noise ratio (S/N) of 3. Good linearity was obtained in the range from 55 fmol (S/N 3) to 500 fmol (r²=0.9968) for SA by using LC/MS with the SRM mode. We also show that the method is useful to analyze SA level in the cytosol of mastocytoma cells, which were pretreated with SA. These results suggest the applicability of this method for the highly sensitive determination of SA in the mammalian tissues and cells.