Background. Saliva is a popular biospecimen for the measurement of hormones, yet fluctuations in hormone levels limit the extent to which saliva can address focusing on basal or long-term levels. Hair steroid assays return basal hormonal levels by collapsing across short-term hormonal variability, including menstrual cyclicity. Here we sought to validate a hair bioassay methodology that can capture stable estradiol levels from both human and monkey hair samples. Methods. Three projects were involved to examine hair-saliva correspondence and estradiol stability in hair. Project 1. Saliva samples were collected once per week for two cycles in 11 emerging adult women. Hair samples were collected at the end of each menstrual cycle and were segmented by 1 cm for the first 4 cm to reflect the past four serial months’ hormone levels. Project 2. Hair samples collected from 23 adolescent participants (Mage = 14.1, 56.5% female) were cut to three 1.5 cm segments from the scalp end. Project 3. Two hair samples were collected from two adjacent skin areas on each monkey (N = 8, 75% males). Whole hair samples were sheared and used for assay without segmentation. Hair biospecimens were processed using a double-extraction protocol validated in this study, then assayed using commercially-available enzyme-immuno-assays for estradiol. Results. Project 1. Hair estradiol concentrations were significantly associated with averaged saliva estradiol levels (r = 0.77, p< .05). Estradiol levels in two consecutive segments were significantly associated (1st vs. 2nd: r = .63, p < .01; 2nd vs. 3rd: r = .49, p < .05; 3rd vs. 4th: r = .53, p < .05). Project 2. Estradiol concentrations were significantly correlated between the first two successive hair segments from the scalp end (r = .69, p < .01). Project 3. Estradiol levels in the two hair samples from each monkey were significantly correlated (r = .66, p < .05).Discussion. Results suggest hair captures valid and reliable average estradiol concentrations using a double-extraction protocol that is applicable for both human and monkey hair. The measurement of hair provides valuable information on individual differences in average estradiol levels across months. Results also indicate that it is feasible and reliable to collect the first 2-3 centimeters of studies in which basal estradiol levels in the past 2 to 3 months are of interest as a stable hormonal index for different species.