SummaryMycoplasma infection leads to false and non-reproducible scientific data and poses a risk to human health. Despite strict guidelines calling for regular mycoplasma screening, there is no universal and widely established standard procedure. Here, we describe a reliable and cost-effective PCR method that establishes a universal protocol for mycoplasma testing. The applied strategy utilizes ultra-conserved eukaryotic and mycoplasma sequence primers covering by design 92% of all species in the six orders of the classMollicuteswithin the phylum Mycoplasmatota and is applicable to mammalian and many non-mammalian cell types. This method can stratify mycoplasma screening and is suitable as a common standard for routine mycoplasma testing.