Background: In non-pregnant populations, pancreatic stone protein (PSP) has been reported to have a higher diagnostic performance for identifying severe inflammatory and infectious disease than other established biomarkers. Objective: To generate reference values for serum PSP in pregnancy and compare them to the values of the general healthy population. Design: A prospective cohort study. Setting: A single center. Population: Healthy women with singleton and multiple pregnancies. Methods: This is a prospective single-center cohort study. Between 2013 and 2021, samples of 5 mL peripheral blood were drawn from 440 healthy pregnant women. Therein, 393 cases were singletons and 47 were multiple pregnancies. Serum PSP levels were measured by specific enzyme-linked immunosorbent assay. The main outcome measures were serum PSP level (ng/mL) reference values in healthy pregnant women. Results: The mean PSP reference values in women with singleton pregnancies were 7.9 ± 2.6 ng/mL (95% CI; 2.69–13.03 ng/mL). The PSP values in women with multiple pregnancies (9.17 ± 3.06 ng/mL (95% CI; 3.05–15.28 ng/mL)) were significantly higher (p= 0.001). The PSP values in the first trimester (6.94 ± 2.53 ng/mL) were lower compared to the second (7.42 ± 2.21 ng/mL) and third trimesters (8.33 ± 2.68 ng/mL,p= 0.0001). Subgroup analyses in singletons revealed no correlations between PSP values, maternal characteristics, and pre-existing medical conditions. Conclusion: The PSP values in healthy pregnant women (4–12 ng/mL) were in the range of the reference values of the general healthy population (8–16 ng/mL). This insight blazes a trail for further clinical studies on the use of PSP as a potential novel biomarker for the early detection of pregnancy-related diseases such as chorioamnionitis.