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Summary: The alarming problem of meat adulteration emphasises the demand for accessible analytical approaches for food regulatory agencies to detect and, specially, to measure altered meat fractions. This study proposes a novel cross‐species triplex droplet digital polymerase chain reaction (ddPCR) assay to simultaneously identify and quantify the ratios of pork/beef meat fractions from a total DNA content, including processed and autoclaved meat, without requiring a standard, achieving high sensitivity with a limit of quantification estimated at 0.1% (w/w) and a limit of detection down to 0.01% (w/w). A single copy nuclear gene, β‐actin, was employed as a target, accompanied with myostatin gene as a cross‐species target to quantify the meat background. The duplex assay provided a simultaneous quantification of pork and myostatin, whereas the triplex assay was able to detect pork, beef and myostatin with a decrease of technical error, cost and time. [ABSTRACT FROM AUTHOR]