The MycoQSearch™ Plus Mycoplasma Detection Kit is a hydrolysis probe-based qPCR assay designed for the rapid and accurate detection of mycoplasma contamination in cell culture and biopharmaceutical manufacturing processes. The assay enables broad-spectrum detection of more than 200 mycoplasma species, including Acholeplasma, Spiroplasma, and Ureaplasma. The test procedure has been validated for sensitivity, specificity, and robustness in accordance with European Pharmacopoeia 2.6.7, providing reliable and reproducible results. In addition, comparability studies were carried out, supporting its successful implementation as an alternative method. Furthermore, the kit incorporates a dual-probe system to enable identification of cross-contamination derived from positive control DNA.