Folates play a key role in the prevention of neural tube defects in newborns. Thus, it is important to reliably determine the bioavailability of folates from various foods. Accurate analytical methods are essential for quantifying blood-folates, especially in human studies. Here, we present the development and validation of a sensitive method using stable isotope dilution liquid chromatography coupled with mass spectrometry for determining various folates in plasma. Moreover, this study reports the applicability of the developed method to a human pilot study using strawberries as a test food. Validation of the assay revealed the precision, sensitivity, and accuracy of the method in determining the predominant 5-methyltetrahydrofolate in plasma. This method was also applicable for the screening of individual folate status using finger prick blood and for monitoring the post-absorptive plasma-concentration curve. Moreover, the human study revealed a high recovery of strawberry folates with a calculated relative bioavailability of 96.2%. Thus, the developed method enables prospective bioavailability studies. This work also confirmed, via human studies, that strawberries are a rich and natural source of folates that are available for human metabolism.