Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease. Tofacitinib is a Janus Kinase inhibitor licensed for the treatment of RA that, unlike biologic anti-rheumatic drugs, is administered orally, but studies of long-term treatment adherence rates are lacking. The measurement of adherence, however, is challenging and there is currently no gold standard test for adherence. Here, we developed a novel HPLC MS/MS assay for the quantification of tofacitinib. The assay demonstrated a LLOQ for tofacitinib of 0.1 ng ml-1, within run accuracy was 81-85% at LLOQ and 91-107% at all other levels. To investigate the ability of the assay to detect adherence, tofacitinib was measured in a random selection of serum samples (n = 10) of tofacitinib treated RA patients who self-reported adherent behaviour. The assay measured tofacitinib in all samples above the LLOQ demonstrating the potential of the assay to sensitively measure biochemical adherence in real-world patient samples. This method for detection of adherence has the potential to be a more objective measure that could be used in the future in the clinic but will require further studies to explore factors that may influence measurement of drug levels, such as clinical characteristics of patients.