We herein report a universal calibration curve for the UV-Visible spectrophotometric determination of the concentration of polymerizable dyes (C dye ) in solution. The equation for the calibration curve is ∑ ( ) , in which m is the number of dye species in solution, both monomeric and aggregates, I F is the instrumental factor that takes values around 1, L is the path length, are the absorbance values at λ i , which are the characteristic wavelengths of maximum absorption for all dye species, and β i are coefficients of proportionality that can be evaluated either with the molar attenuation coefficients of the different dye species (if known) or via error minimization. The method has been successfully applied to construct a calibration curve of methylene blue in water which is applicable in a wide range of methylene blue and chloride concentrations, regardless of both the aggregate concentration distribution and the temperature. In addition, it was found that the molar fractions of each MB species in solution could be fairly approximated by means of simple algebraic expressions in the A 650 /A 607 absorbance ratio.