Multi-Energy Calibration (MEC) was recently proposed as an innovative analytical method to be used in efficient and accurate quantitative analysis based on atomic spectroscopy. Here, the applicability of the MEC method for quantifying molecular species using UV-Vis and fluorescence measurements was evaluated for the first time. UV-Vis and fluorescence spectra of the analytical solutions of methylene blue and eosin-methylene blue in two different solvents (distilled-deionized water and methanol) were collected. MEC showed high precision and sensitivity for determining the analyte concentration, providing similar limit of detection and quantification when compared with conventional analytical methods, such as external standard calibration and standard additions. Therefore, the present study has shown that MEC can be successfully applied for quantifying molecular species in a simple and efficient way accounting UV-Vis and fluorescence spectroscopy.