Gonadal-derived inhibins are essential factors in mammalian reproduction, negatively regulating pituitary production of follicle stimulating hormone (FSH). Interestingly, declines in inhibin levels across the menopause transition do not only correlate with an increase in FSH, but also a rapid decrease in bone mass. Therefore, inhibins have been touted as potential therapeutics for osteoporosis in post-menopausal women. However, as heterodimeric proteins of α- and β (βA or βB)-subunits, inhibins are difficult to produce recombinantly, they are poorly processed to their mature bioactive forms and their expression is always accompanied by production of activins (β-subunit homodimers); the proteins they antagonise. In this study, we developed the methodology to circumvent activin interference in inhibin production and bioactivity. Initially, the cleavage sites between the pro- and mature domains of the α- and βA-subunits were modified to ensure complete processing. These modifications led to a marked increase (9-fold) in the levels of bioactive inhibin A and a striking decrease (12.5-fold) in mature activin A production. Significantly, using targeted in vitro mutagenesis, we were able to disrupt the formation of activin β/β homodimers, enabling an inhibin α/β heterodimer production bias. Next, a single point mutation (M418A) was incorporated into the βA-subunit, which reduced residual activin activity ~100-fold and, in so doing, increased inhibin bioactivity 8-fold. We also showed that inhibin A non-covalently associated with its prodomain was more potent (~20-fold) than mature inhibin A in specific in vitro bioassays, indicating an important role of the prodomain in inhibin bioactivity. In conclusion, the production of potent inhibin analogues in the virtual absence of activin activity will greatly facilitate the investigation of the therapeutic potential of these gonadal hormones on bone and other tissues.