Human milk (HM) contains numerous non-nutritive bioactive factors, amongst which the peptide hormone insulin. HM insulin has been suggested to accelerate intestinal maturation, thereby promoting feeding tolerance. Therefore, recombinant human insulin for enteral administration has been developed which might serve as supplement to HM or formula for preterm infants. However, the natural course of the HM insulin concentration directly following delivery is unknown, which hampers the development of dosage schedules in clinical trials. The aim of this study was to validate a method for insulin determination in small volumes of HM, and to assess the stability of HM insulin. The results showed that the HM insulin concentration can be measured rapidly and reliably by using an automated immunoassay. In addition, HM insulin is stable at 4 °C for at least 72 h, at room temperature for a maximum of 12 h, at −20 °C for at least 2.5 years, and during at least five freeze-thaw cycles.