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Background: Measurement of hypoglycin A (HGA) and its toxic metabolite, methylenecyclopropylacetic acid (MCPA), in equine serum confirms a diagnosis of atypical myopathy (AM), a pasture‐associated toxic rhabdomyolysis with high mortality linked to the ingestion of Acer trees plant material. Supportive diagnostic tests include plasma acyl‐carnitine profiling and urine organic acid testing, but these are not specific for AM. Previously reported HGA and MCPA analytical techniques used liquid chromatography–mass spectrometry (LC‐MS) with a derivatising step, but the latter prolongs testing and increases costs. Objectives: To develop a rapid LCMS method for detection of serum and tissue HGA and MCPA that enables expedited diagnosis for horses with AM. Study design: Analytical test validation. Methods: Validation parameters to industry standards using as criteria precision, accuracy, linearity, reproducibility and stability in analyte‐spiked samples were calculated on 9‐calibration points and 3 different validation concentrations in both serum and muscle tissue. Results: The test was successfully validated for the detection of HGA and MCPA‐carnitine in equine serum and muscle. Test linearity was excellent (r2 =.999), accuracy was very good for both analytes (93%‐108%), precision did not exceed 10% coefficient of variation and reproducibility met the requirements of the Horwitz equation. Stability was unaffected by storage at a range of temperatures. Main limitations: The spectrum of the tested analytes was limited to only two relevant analytes in favour of a quick and easy analysis. Linearity of the muscle method was not evaluated as calibration curves were not produced in this matrix. Conclusion: We report an optimised, simplified and validated method for detection of HGA and MCPA‐carnitine in equine serum and muscle suitable for rapid diagnosis of suspected AM cases. The serum‐based test should also enable risk assessment of toxin exposure in cograzing horses and assessment of horses with undiagnosed myopathies, while the tissue detection test should help to confirm cases post‐mortem and to determine toxin distribution, metabolism and clearance across different tissues. [ABSTRACT FROM AUTHOR]