A HPLC-UV/Vis method using a two-column strategy with a switching valve for on-line sample clean-up was developed for the determination of cyanocobalamin (CN–Cbl/Vitamin B12) in multivitamin dietary supplement tablets. The method uses two columns: an Agilent Zorbax C8 (150 mm × 4.6 mm, 5 μm particle size) reversed-phase column and a Waters Symmetry C18 (150 mm × 4.6 mm, 5 μm particle size) reversed-phase column. Chromatographic separation was achieved using a programmed gradient mobile phase consisting of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile. Because of the low levels of Vitamin B12 in the samples, large injection volumes, and thus much interfering material, must be used to exceed the limit of quantitation (LOQ) by UV detection. A switching valve was used to divert most of these early eluting interfering materials to waste, effecting on-line sample clean-up without excessive sample preparation steps. The recovery of CN–Cbl in the method was 99.5% and the LOQ was 10 ng per injection. The method was successfully applied to the analysis of the NIST SRM 3280 multivitamin/multimineral dietary supplement tablet. The method is specific, precise, and accurate for the intended use. Compared to off-line sample clean-up procedures, it offers the advantage of being easier, more economical, and less time-consuming.