Histamine is an important biogenic amine involved in regulating numerous physiological and pathophysiological processes in humans and animals. To date, there have been very few studies focused on developing and validating sensitive liquid-chromatography–tandem mass spectrometric (LC–MS/MS) assays capable of quantitative trace level histamine analysis in biological matrices. In the present study, a rapid and sensitive LC–MS/MS assay, amenable to high throughput analysis was developed and validated to characterize in vitro and in vivo histamine release. The LC–MS/MS procedure incorporating deuterium labeled internal standards provides rapid resolution of histamine with excellent sensitivity, precision, and accuracy. Histamine eluted at 1.5 min and was well separated from endogenous plasma peaks. The total run time of the assay was 8.0 min. A linear (r2 ≥ 0.99) instrument response over the entire concentration range of 1.0–1000 ng/mL was observed. Excellent accuracy (error ± 3.4%) and precision (CV ± 10%) of the assay was demonstrated, with the lower limit of quantitation (LLOQ) at 15.6 ng/mL. The validated LC–MS/MS assay was applied to determine histamine release in both in vitro and in vivo models. Peritoneal mast cells treated with prototypical degranulating agents (Compound 48/80 and Teicoplanin) showed that the two chemicals caused approximately 40% histamine release. In rats, using this assay, basal histamine plasma levels were typically under 100 ng/mL. Treatment with an agent suspected of causing anaphylactic type reactions resulted in plasma histamine levels to increase above 3000 ng/mL. The LC–MS/MS assay presented in this study can be applied to further characterize the physiological and pathophysiological role of histamine release in complex in vitro and in vivo models. Importantly, the LC–MS/MS assay may be useful in assessing active pharmaceutical ingredient-mediated degranulation and anaphylaxis as part of either a pre-market or a post-market assessment of drug products.