Early life exposure to folate has long lasting effects on development and health. Newborns obtain part of their folate from maternal milk. Studies on health effects of milk folate require rapid, affordable and reliable measurements in large numbers of samples from cohort studies. Recently, a competitive chemiluminescence assay for quantification of folate has become available for automated diagnostic measurement of folate in human serum or plasma. We tested if this method (“FOLA” from Siemens Healthcare) could also be used for human milk. To minimize interference and matrix effects, samples had to be skimmed, diluted seven times with demineralized water, and heated for 5 min at 90 °C. Folate could thus be measured in a linear range between 8.4 and 111.7 nM, with recoveries for the most relevant form, 5-methyltetrahydrofolate (5-MeTHF), of 96%–107%. Results were comparable to those with a recently validated Liquid Chromatography/Mass Spectrometry method (Y = 0.998X − 0.2; R² = 0.807). The FOLA method was subsequently used for samples from the LIFE Child cohort in Germany, providing first data of breast milk folate in this country (range: 6.2–100.7 nM). This technique could indeed prove useful for large cohorts with multiple samplings.