Deltamethrin, a well-known type 2 synthetic pyrethroid insecticide, is a widespread environmental toxicant. It has potential to accumulate in body fluids and tissues due to its lipophilic characteristics. The immune system is among the most sensitive targets regarding toxicity of environmental pollutants. Various methods are available in the literature to analyze deltamethrin (DLM) concentration in plasma and tissues, but regarding the immune organs, only one gas chromatography–tandem mass spectrometry (GC–MS/MS) method (on spleen tissues) has been reported. In the present investigation, a rapid and sensitive high-performance liquid chromatography (HPLC) method has been developed and validated to determine DLM concentration in plasma, thymus, and spleen using zaleplone as an internal standard. Liquid chromatography (LC) separation is performed on an Agilent Zorbax® C8 column (250 mm × 4.6 mm, i.d., 5 μm) with isocratic elution using a mobile phase consisting of acetonitrile–5 mM KH2PO4 (70:30, v/v) at a flow rate of 1 mL min−1. The lower limit of quantification (LLOQ) for DLM is 10 ng mL−1 (plasma, thymus, and spleen). The method has been validated in terms of establishing linearity, specificity, sensitivity, recovery, accuracy, and precision (intra- and inter-day) and stabilities study. This validated method was successfully applied to a pharmacokinetic and tissue distribution study of DLM in mice.