Folate deficiency is a widespread nutritional issue, and biofortifying dairy products through lactic acid bacteria (LAB) is a promising strategy to enhance natural folate levels. This study aimed to develop a reliable method for selecting Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus strains with enhanced folate production for use as functional starter cultures. Initially, a traditional microbiological assay (MA) was used to measure folate production in 36 LAB strains isolated from fermented milks. Due to MA's limitations, an untargeted and semi-quantitative method combining ultra-high-performance liquid chromatography (UHPLC) with high-resolution mass spectrometry (HRMS) was developed for a more comprehensive folate screening. The MA showed higher folate production in S. thermophilus strains (309-639 µg/L) compared to L. delbrueckii subsp. bulgaricus (up to 48 µg/L). Subsequently, nine selected LAB strains were further analyzed using the UHPLC-HRMS approach, which enabled the identification and semi-quantification of six folate metabolites, namely dihydrofolate, tetrahydrofolate (THF), 10-formyl-THF, 5,10-methenyl-THF, 5,10-methylene-THF, and 5-methyl-THF. Lab-scale yogurt production using the top-performing strains, as identified through the HRMS method, demonstrated an increase in folate content over a 14-day shelf life. These findings revealed the potential of UHPLC-HRMS as a high-throughput alternative method for folates detection, offering a promising tool for screening folate-enhanced LAB strains for biofortification.