Background: Available measurements of women's sexual function do not account for different sexual orientations; rather, instruments have been developed using heterosexual samples. The Female Sexual Functioning Index (FSFI) is a widely used instrument, applicable for sexually active or inactive women. We apply the FSFI to a sample of women who have or prefer women as sexual partners, defined as sexual minority women, and who vary with respect to their sexual activity. Methods: A modified version of the FSFI was used in a sample of sexual minority women. Statistical analyses focused on examining associations between FSFI responses of no sexual activity and women's characteristics. Results: Partner status and sexual frequency was significantly associated with reporting no sexual activity on the FSFI. A revised scoring of the FSFI allows for the use of this instrument among women who vary on sexual frequency and partner status, without biasing their scores towards sexual dysfunction. The desire subscale is independent of sexual frequency, partner status, and sexual orientation. Conclusions: The modified wording of the FSFI and its revised scoring allow for the use of this instrument among sexual minority women. A separate reporting of the desire subscale will generate reliable and valid assessments of sexual minority women's sexual functioning.