Methylene blue is a heterocyclic dye with a wide range of use in biology and chemistry. Purity of dyes rarely exceed 70% and separation of methylene blue and related impurities is required. Two alternative methods for purity analysis were developed on Primesep C and Primesep D mixed-mode HPLC columns. Primesep C is a reversed-phase cation-exchange (anion-exclusion column) column and Primesep D is reversed-phase anion-exchange (cation-exclusion) column. Various buffers can be used for the method. Depending on the buffer, method can be compatible with UV, LC/MS and prep chromatography. Method can be used for prep separation of methylene blue and related impurities.