Ankylosing spondylitis (AS) is a chronic inflammatory disease. In many inflammatory diseases, increased production of pro-inflammatory cytokines is associated with an increase in oxidative stress mediators. Thiol/disulfide homeostasis is a marker for oxidative stress. The aim of this study was to examine the dynamic thiol/disulfide homeostasis in AS. Sixty-nine patients with AS and 60 age- and sex-matched controls were included in the study. The Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and visual analogue scale (VAS) were used to determine the disease activity. Native thiol, total thiol, and disulfide levels were measured with a novel automated method recently described by Erel and Neselioglu. The aforementioned method is also optionally manual spectrophotometric assay. The total thiol levels were significantly lower in the AS group compared with the control group (p = 0.03). When the patients were divided into active (n = 35) and inactive (n = 34) subgroups using BASDAI scores, the native plasma thiol and total thiol levels were significantly lower in the active AS patients compared to the inactive AS patients (p = 0.02, p = 0.03 respectively). There was a negative correlation between the plasma native thiol levels and VAS, BASDAI scores. Thiol/disulfide homeostasis may be used for elucidating the effects of oxidative stress in AS. Understanding the role of thiol/disulfide homeostasis in AS might provide new therapeutic intervention strategies for patients.