Nipple aspirate fluid is the physiological biofluid lining ductal epithelial cells. Historically, cytology of nipple fluid has been the gold standard diagnostic method for assessment of ductal fluid in patients with symptomatic nipple discharge. The role of biomarker discovery in nipple aspirate fluid for assessment of asymptomatic and high-risk patients is highly attractive but evaluation to date is limited by poor diagnostic accuracy. However, the emergence of new technologies capable of identifying metabolites that have been previously thought unidentifiable within such small volumes of fluid, has enabled testing of nipple biofluid to be re-examined. This review evaluates the use of new technologies to evaluate the components of nipple fluid and their potential to serve as biomarkers in screening.