Purpose <p>To review established and emerging biochemical pituitary biomarkers, examining how biological sex influences their concentrations and diagnostic interpretation.</p> Methods <p>We performed a literature review of recent studies and guidelines on all pituitary hormone axes, emphasizing sex-specific differences, assay variability, and new biomarkers of pituitary function.</p> Results <p>Sex significantly modulates all major pituitary axes. For example, women experience cyclical or estrogen-driven variations in gonadotropins, cortisol binding, and growth hormone (GH) secretion, whereas men have more stable hormonal patterns. These differences necessitate sex-specific reference intervals in contexts such as reproductive hormones, pregnancy, and IGF-I concentrations to ensure accurate interpretation. Additionally, emerging biomarkers offer new diagnostic tools: Soluble α-Klotho (sαKL) is a GH-responsive protein that is elevated in acromegaly and shows relatively modest sex-related variation in healthy adults. Copeptin, a stable surrogate for arginine vasopressin (AVP), measured in stimulation tests now outperforms the classical water deprivation test for differentiating AVP-deficiency, with markedly higher diagnostic accuracy. Furthermore, neurophysin I – an equimolar fragment co-released with oxytocin – has been validated as a robust proxy for oxytocin secretion, rising in parallel with oxytocin during stimulation and thus overcoming prior analytic challenges.</p> Conclusion <p>Recognizing and adjusting for sex-specific differences in pituitary biomarkers is essential to improve diagnostic precision and to ensure more equitable, personalized care in pituitary disorder management.</p>