SIGNIFICANCE: Methylene blue (MB) is an exogenous contrast agent that has the potential to assist with visualization and penetration challenges in photoacoustic imaging. However, monitoring the local concentration between MB and endogenous chromophores is critical for avoiding unnecessary MB accumulations that could lead to adverse effects such as hemolysis when exposed to increased dose and photodamage when exposed to high laser energies. AIM: We developed a modified version of a previously proposed acoustic-based atlas method to estimate concentration levels from a mixture of two photoacoustic-sensitive materials after two laser wavelength emissions. APPROACH: Photoacoustic data were acquired from mixtures of 100-μM MB and either human or porcine blood (Hb) injected in a plastisol phantom, using laser wavelengths of 710 and 870 nm. An algorithm to perform linear regression of the acoustic frequency response from an atlas composed of pure concentrations was designed to assess the concentration levels from photoacoustic samples obtained from 11 known MB/Hb volume mixtures. The mean absolute error (MAE), coefficient of determination (i.e., R2), and Spearman's correlation coefficient (i.e., ρ) between the estimated results and ground-truth labels were calculated to assess the algorithm performance, linearity, and monotonicity, respectively. RESULTS: The overall MAE, R2, and ρ were 12.68%, 0.80, and 0.89, respectively, for the human Hb dataset and 9.92%, 0.86, and 0.93, respectively, for the porcine Hb dataset. In addition, a similarly linear relationship was observed between the acoustic frequency response at 2.3 MHz and 870-nm laser wavelength and the ground-truth concentrations, with R2 and | ρ | values of 0.76 and 0.88, respectively. CONCLUSIONS: Contrast agent concentration monitoring is feasible with the proposed approach. The potential for minimal data acquisition times with only two wavelength emissions is advantageous toward real-time implementation in the operating room.