Allergies are highly complex disorders with clinical manifestations ranging from mild oral, gastrointestinal, recurrent wheezing, and cutaneous symptoms to life-threatening systemic conditions. The levels of arachidonic acid, eicosanoids, histamine, organic acids and valine are considered to have a variety of physiological functions in connection with allergies. In this research, we have developed a RP-LC/MS method to separate and quantitate six different potential endogenous biomarkers, including leukotrieneB4 (LTB4), prostaglandinD2 (PGD2), arachidonic acid (AA), histamine (HI), lactic acid (LA) and valine (VAL), from serum of rats with ovalbumin (OVA)-induced allergy and normal rats, and the discrepancies between the model group and the control group were compared. The separation was performed on a Prevail C18 column (250 mm × 4.6 mm, 5 μm) with a gradient elution of acetonitrile with 0.1% formic acid (v/v) and 10 mM ammonium formate (adjusted to pH 4.0 with formic acid) at a flow rate of 0.5 mL min−1 The method was validated and shown to be sensitive, accurate (recovery values 76.16–92.57%) and precise (RSD < 10% for all compounds) with a linear range over several orders of magnitude. The method was successfully applied to rat serum and shown to be indicative of the endogenous levels of biomarkers within the rat body. The analysis of the biomarkers can provide insight into the allergic mechanisms associated with related diseases.