Mycoplasma genitalium is an emerging global health threat, due to an alarming rise in antimicrobial resistance. Although individualised treatment approaches have been successfully adopted for macrolides, treatment is complicated by rising rates of macrolide resistance and limited alternative treatments. Fluoroquinolones are another mainstay treatment for M. genitalium; however, individualised fluoroquinolone therapy has not yet been explored, partly due to the scarcity of commercial assays and lack of confidence in specific mutations associated with resistance. We conducted a comprehensive literature review the genotypic and phenotypic determinants of fluoroquinolone resistance, and coupled with contemporary Australian clinical data. Based on this strong data, we propose a clear role for the development of a molecular test that incorporates markers to detect both ParC wild-type, and the most common mutation S83I, as respective predictive markers of fluoroquinolone susceptibility and resistance. Based on this, we have developed a range of assays that detect both ParC wild-type and S83I, either as TaqMan probe technologies or novel probe-based melting curve assays, to characterise these key markers of fluoroquinolone susceptibility and resistance. These assays can greatly improve first-line antimicrobial selection and stewardship and rationalise test of cure for this clinically challenging pathogen, which is extremely valuable in the care of patients with M. genitalium infections.