The menopause transition is associated with an increased risk of depressed mood. Preliminary evidence suggests that increased sensitivity to psychosocial stress, triggered by exaggerated perimenopausal estradiol fluctuation, may play a role. However, accurately quantifying estradiol fluctuation while minimizing participant burden has posed a methodological challenge in the field. The current pilot project aimed to test the feasibility of capturing perimenopausal estradiol fluctuation via 12 weekly measurements of estrone-3-glucuronide (E1G), a urinary metabolite of estradiol, using participant-collected urine samples in 15 euthymic perimenopausal women ages 45-55 years. Furthermore, it aimed to correlate E1G fluctuation (standard deviation across the 12 E1G measurements) with weekly mood and cardiovascular, salivary cortisol, and subjective emotional responses to the Trier Social Stress Test (TSST) at weeks 4, 8, and 12. Protocol acceptability and adherence was high; furthermore, E1G fluctuation was positively associated with anhedonic depressive symptoms and weekly negative affect. E1G fluctuation was also associated with increased heart rate throughout the TSST as well as higher levels of rejection, anger, and sadness. E1G fluctuation was not significantly associated with TSST blood pressure or cortisol levels. This study suggests a feasible method of assessing estradiol fluctuation in the menopause transition and provides support for the hypothesis that perimenopausal estradiol fluctuation increases sensitivity to psychosocial stress and vulnerability to depressed mood.