Background & aims: Donor human milk (DHM) is recommended as the first alternative for preterm infants if their mother's own milk is not available or if the quantity is not sufficient. The most commonly used technique to eliminate microbial contaminants in DHM is holder pasteurization (HoP). However, the heating process during HoP partially destroys milk bioactive factors such as insulin. Therefore, innovative techniques have been developed as alternatives to HoP. The objective of this study was to determine the effect of HoP, high-temperature–short-time (HTST), thermoultrasonication (TUS), ultraviolet-C irradiation (UV-C), and high-pressure processing (HPP) on the insulin concentration in DHM. Methods: Milk samples from 28 non-diabetic mothers were collected. The milk samples were aliquoted and either left untreated or treated with HoP (62.5 °C; 30 min), HTST (72 °C; 15 s), TUS (60 W; 6 min), UV-C (4863 J/L), or HPP (500 MPa; 5 min). Results: The mean insulin concentration in untreated milk was 79 ± 41 pmol/L. The mean insulin retention rate was 67% for HoP, 78% for HTST, 97% for TUS, 94% for UV-C, and 106% for HPP. The mean insulin concentration in milk treated with HoP was significantly lower compared to untreated milk (p = 0.01). Conclusion: TUS, UV-C, and HPP preserve insulin in DHM. The insulin concentration in DHM is affected to a larger extent by HoP than by HTST. These results indicate that TUS, UV-C, and HPP may serve as alternatives to HoP.