Background: The determination of cyanocobalamin (vitamin B12) and biotin has always been challenging because of the lack of a chromophore for biotin and trace level input for vitamin B12 in supplements. Microbiological assay methods are currently used for quantitation. However, these methods are time consuming, may lack specificity, and have high imprecision. Objective: Our laboratory developed and validated an LC method for the simultaneous quantitation of vitamin B12 and biotin. Methods: This LC method uses a single quadruple mass analyzer to detect biotin and vitamin B12 at m/z 245.10 and m/z 678.29, respectively. Results: The mass analyzer allows for low limits of quantitation (biotin: 1 ng/mL; vitamin B12: 4 ng/mL). Precision results showed that injections are repeatable without the use of an internal standard (RSD < 5%). Single analyst (n = 5: RSD < 3%), within lab (n = 10: RSD < 8%), and multilab (n = 20: RSD < 13%) precision results were also much better than those reported by microbiological assay methods. Linearity was evaluated between 92.00 ng/mL and 9200 ng/mL (R² 09916) for biotin and between 4.846 ng/mL and 484.6 ng/mL (R² 0.9999) for vitamin B12. The method is accurate between 20 ng/mL and 60 ng/mL for vitamin B12 and between 400 ng/mL and 1200 ng/mL for biotin. Conclusions: The results show a simple, accurate, and precise method for the quantitation of vitamin B12 and biotin. Highlights: This work demonstrates that single quadrupole mass analyzers can be successfully used to quantify trace level analytes in quality control laboratories.