Prostanoids, derivatives of arachidonic acid, are involved in inflammation and immune reactions. To understand the role of prostanoids produced by diverse immune cells, a highly sensitive quantitation method for prostaglandin E2 (PGE2), prostaglandin D2 (PGD2), 6-keto prostaglandin F1α (6-keto PGF1α), prostaglandin F2α (PGF2α), and thromboxane B2 (TXB2) by means of nano-liquid chromatography-tandem mass spectrometry has been developed. It was validated according to the guidelines of the Food and Drug Administration (FDA) in terms of linearity, precision, accuracy, recovery, stability, and lower limit of quantitation (LLOQ). The LLOQ were 25 pg/mL in the injected solution (75 fg on column (o.c.)) for PGE2 and PGD2 and 37.5 pg/mL (112.5 fg on column) for 6-keto PGF1α, PGF2α, and TXB2, respectively. It was successfully applied to murine mast cells isolated from paws after zymosan injection and to CD4(+) and CD8(+) T lymphocytes from blood of sensitized versus non-sensitized mice in context of a delayed type hypersensitivity model. About 5,000 (T cells) to 40,000 (mast cells) cells were sufficient for quantitation. In the mast cells, the production of PGE2 increased at a significantly higher extent than the synthesis of the other prostanoids. The T lymphocytes did not show any difference in prostanoid production, no matter whether they were obtained from sensitized mice or non-sensitized mice.