The purpose of this study was to investigate the prevalence of antinuclear antibody (ANA) positivity in a cohort of patients with hidradenitis suppurativa (HS), and to assess the frequency of seroconversion during treatment with tumor necrosis factor (TNF)-α inhibitor therapy. This prospective study was conducted through the Wound Etiology and Healing (WE-HEAL) Study. Immunofluorescence ANA testing was performed at baseline, and repeated when clinically indicated. ANA titers of ≥1:160 were considered positive. Data were collected on demographics and disease activity scores including the Hurley stage, the HS Sartorius score (HSS) and the active nodule (AN) count. At the time of data lock, 73 patients with a confirmed diagnosis of HS were enrolled, and four (5.4%) had baseline positive ANA. None of the patients had clinical evidence of systemic lupus erythematosus or other autoimmune diseases. There were no significant differences in demographics, baseline HSS (43.25±47.55 compared to 59.48±56.67, P=0.58) or AN count (3.25±3.20 compared to 3.45±2.36, P=0.87) in the ANA positive group. Of the 69 patients who were ANA negative at enrollment, 31 (45%) received TNF-α inhibitor therapy. During follow up, one patient developed drug-induced lupus secondary to TNF-α inhibitor use. Additionally, one patient seroconverted to ANA positive without sequelae and one patient developed drug-induced hepatitis secondary to TNF-α inhibitor use. The prevalence of baseline ANA positivity in this HS population was similar to that seen in the general population (5.4%). The rate of seroconversion and drug-induced complications in this population were low.