Milk is one of the most widely consumed animal‐derived food due to its nutritional properties. It is mainly derived from animals, which are being treated with anti‐tuberculosis (anti‐TB) drugs for disease treatment and prevention, hence, these may excrete from animals through milk, thus, pose a threat to consumers. An analytical method based on automated micro‐solid phase extraction coupled to liquid chromatography‐triple quadrupole mass spectrometry was developed for the analysis of anti‐TB drugs in milk samples. The developed method was validated and estimated measurement uncertainty for its applicability to routine analysis. The method linearity was in the range of 25–1000 ng/ml with a coefficient of determination between 0.994 and 0.999. The limit of detection and limit of quantification were in the range of 1.342–9.606 and 4.469–31.98 ng/ml, respectively for anti‐TB drugs. The percent recovery was found to be in the range of 82.29%–111.84%, and the expanded uncertainty of the proposed and validated method was observed to be in the range of 2.94%–28.1%. The present method has been able to analyze 16 anti‐TB drugs in different milk samples and was found to be quick, rapid, high‐throughput, cost‐effective, precise and accurate, and useful for routine testing of anti‐tuberculosis drugs in milk samples.

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