Background: The main cause of adrenal insufficiency (AI) in paediatric patients is prolonged treatment with corticosteroids. Determination of plasma cortisol (PC) during ACTH test is the most used adrenal function indicator in clinical practice. However, determination of salivary cortisol (SC), a simple test especially useful in children in order to avoid invasive procedures, can be used as an alternative technique for the diagnosis of adrenal disease. Methods: A two-year prospective study (January 2014-January 2016) in paediatric patients (2-18 years of age) treated with corticosteroids for more than fifteen days, who were investigated for suspected AI. Low-dose ACTH test was used to determine adrenal function and samples for SC and PC were obtained simultaneously in basal situation and during the test (at 30, 60 and 90 minutes). Results: 230 samples (118 PC-112 SC) of 30 studies belonging to 20 patients (4 males), mean age 10.93 years ± 3.69 SD. Pearson’s correlation coefficient showed a positive correlation between PC and SC (r = 0.618, p < 0.001). All the studies with some determination of PC higher than 18 μg/dL (n = 8) had a SC peak higher than 0.61 μg/dL with a specificity of 66.67% and a sensitivity of 93.94% (ROC analysis). Conclusion: Measurement of SC is a less invasive, easier and quicker test than PC to measure plasma free cortisol levels. In our study, a SC peak in low-dose ACTH test higher than 0.61 μg/dL was able to discriminate patients without AI, and proved to be a useful tool in the initial evaluation of children with suspected AI.Introduction The activation of the hypothalamic-pituitary-adrenal axis in response to critical illness and the resulting release of cortisol from the adrenal cortex are essential to stress adaptation. Adrenal insufficiency (AI) is described as the inability of adrenal glands to produce an appropriate hormonal secretion not only under stress but also in basal situation. Therefore, a low baseline plasma cortisol (PC) (< 5 μg/dL) and a poor cortisol response to stimulation with exogenous adrenocorticotropic hormone (peak < 18 μg/dL) are some of the defining criteria of this condition [1,2]. It is well known that the main cause of AI in paediatric patients is prolonged treatment with exogenous corticosteroids, which is an iatrogenic cause derived from the increasing complexity of paediatric pathologies and the increased use of prolonged high-dose corticosteroid therapy. In clinical practice, adrenal function is usually assessed by the total PC (determined by low-dose ACTH test). This implies the placement of a vascular access which is often a traumatic experience for children. PC includes protein-bound fraction and serum-free cortisol. The latter constitutes the biologically active form of the hormone and is responsible for glucocorticoid activity on peripheral organs. Most of the circulating cortisol is bound to plasma proteins (over 90%), such as cortisol-binding globulin (CBG) and albumin, whereas only about 10% of circulating cortisol is free. Hence, the measurement of plasma-free cortisol level has been considered more representative of adrenal function (especially in critically ill adults and children) [1,2], because some conditions, such as hypoalbuminaemia or hypoproteinaemia (frequent in critically ill patients or in patients with cirrhosis), may lead to misinterpretation of adrenal function with an overestimation of the prevalence of AI. But the direct measurement of free PC is a laboratory-dependent and time-consuming procedure that is not available for routine use. Salivary cortisol (SC) is one of the several indirect methods available to determine free PC [3], as SC levels accurately reflect free PC [4] even in cases of hypoalbuminaemia or CBG abnormality [1,5]. For this reason, in the last years, this technique (SC) has been introduced as a non-invasive tool in the diagnosis of adrenal cortical disorders, for its simplicity and applicability in the paediatric population. However, few studies to date have evaluated the usefulness of SC as a diagnostic method in children with AI. No interactions between exogenous corticoids and SC have been described [6]. The aim of the present study was to assess the usefulness of determining salivary cortisol levels as a diagnostic tool in children with suspected secondary iatrogenic AI.