Updated project metadata. Rapamycin is a naturally derived macrolide antibiotic that demonstrates immunosuppressive, chemotherapeutic, and anti-ageing effects in model organisms and humans. Importantly, Rapamycin and its analogues are currently used in the clinic against various cancers and neurological disorders. Although Rapamycin is widely perceived as a specific allosteric inhibitor of mTOR (mechanistic target of rapamycin), the master regulator of cellular and organismal physiology, its specificity has not been thoroughly evaluated so far. In fact, previous studies in cells and in mice suggested that Rapamycin may be also acting independently from mTOR to influence various cellular functions. Here, we generated a gene-edited cell line, expressing a Rapamycin-resistant mTOR mutant (mTORRR), and assessed the effects of Rapamycin treatment on the transcriptome and proteome of control and mTORRR-expressing cells. Our data reveal a striking specificity of Rapamycin towards mTOR, demonstrated by virtually no changes in mRNA or protein levels in Rapamycin-treated mTORRR cells, even following prolonged drug treatment. Overall, this study provides the first comprehensive and conclusive assessment of Rapamycin’s specificity, with important potential implications for ageing research and therapeutics.