Edible berries are good sources of several phytochemicals. However, the native folate levels in berries are not well known. The structure of native folates contains polyglutamyl chains, which reportedly jeopardize the bioavailability of native folates; further γ-glutamyl hydrolase (GGH) can deglutamylate polyglutamyl chains. In this study we use a validated ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to determine the distribution of polyglutamyl 5-methyltetrahydrofolate (5-CH₃THF-Glu_n), folate vitamers, and total folates in different berries and to monitor changes in their concentration during processing of berries into juice. The pre-boiling treatment was optimized during extraction to stabilize the native polyglutamyl folates profile and facilitate folate extraction, which can replace the traditional di-enzyme treatment process. Additionally, the efficiency of commercially available human recombinant GGH was tested and it was found that a 10 µg GGH/mL extract at a pH of 6 could completely deconjugate polyglutamyl folates into monoglutamyl folates when incubated for 30 min. Pure human recombinant GGH with a higher catalytic efficiency and stable enzymatic properties was better than traditional folate conjugase for this purpose. From experimental analysis, it could be inferred that strawberries and blackberries contained the highest amount of total folates (93-118 µg/100 g), while the total folate contents in blueberries were the lowest. Most of the investigated berries are good to excellent folate sources. This study is the first time that 5-CH₃THF-Glu_n and distribution of folate vitamers in various berries are quantitated. Further, this is the first study to show the application of recombinant pure GGH for the deconjugation of polyglutamyl folates for folate vitamers and total folates analysis.