B-vitamins are essential micronutrients for human health, supporting energy, lipid, glucose, and protein metabolism and reinforcing the immune system. This group of water-soluble compounds includes thiamin (B1), riboflavin (B2), niacin (B3), pantothenic acid (B5), pyridoxine (B6), biotin (B7), folic acid and folate (B9), and cyanocobalamin (B12). Herein, we introduce a novel analytical method of high-performance liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS) employing Orbitrap technology for the simultaneous quantification of all of the B-vitamins in fruit juices (both commercial beverages and freshly squeezed). This method stands out due to its simplified sample preparation, employing direct dilution and filtration of the juices. Chromatographic separation was optimized on a reversed-phase column using gradient elution with water and acetonitrile. Mass spectrometry detection was performed in both positive and negative ionization modes with full-scan MS acquisition to maximize the sensitivity. The method underwent rigorous validation, demonstrating excellent linearity (R² > 0.998), high accuracy (near 100% for juices with matrix-matched calibration), and good precision (RSD < 15%). Matrix effects were investigated and effectively mitigated through matrix-matched calibration curves. The limits of detection and quantification indicated high sensitivity. The validated method was successfully applied to various real fruit juices, including pomegranate, apple, bergamot, citron, and clementine. Our results not only aligned with the label claims for fortified beverages but also revealed higher B-vitamin concentrations in freshly squeezed juices compared to those in their commercial equivalents, highlighting the utility of the developed approach in discerning nutritional quality.