Accurate quantification of efficiency enables rigorous comparison between different photoluminescent materials, providing an optimization path critical to the development of next-generation light sources. Persistent luminescent materials exhibit delayed and long-lasting luminescence due to the temporary storage of optical energy in engineered structural defects. Although these materials have recently gained attention for their potential in a wide range of applications, from smart lighting to in vivo imaging, standard characterization methods do not provide a universal comparison of phosphor performance, making it difficult to assess the efficiency of the different processes involved in afterglow. In this work we establish a protocol to obtain the emission quantum yield of persistent phosphors. We determine the persistent and total luminescence quantum yields by considering the ratio of photons emitted in the afterglow and during charging to those absorbed. The method is first applied to transparent single crystals of the most common persistent phosphors, such as SrAl2O4:Eu2+,Dy3+ and Y3Al2Ga3O12:Ce3+,Cr3+. The versatility of our methodology is then demonstrated by quantifying the quantum yield of a thin film based on ZnGa2O4:Cr3+ persistent luminescent nanoparticles, which are commonly used for in vivo imaging. We confirm the high efficiency of strontium aluminate and reveal a strong dependence of the obtained values on the illumination conditions, highlighting a trade-off between efficiency and brightness, which opens the door to precise optimization of the charging conditions for each material and application. Our results contribute to the development of standard characterization protocols for the analysis of the mechanisms governing afterglow, as well as the assessment of the overall efficiency of the process. Such achievements enable a rigorous comparison of the performance of different persistent materials, allowing for optimization routes beyond the usual trial-and-error approach.This project has received funding from the BBVA Foundation Leonardo Grant for Physics Researchers 2023, and by Grant EUR2023-143467 funded by MICIU/AEI/ 10.13039/501100011033 and by the European Union NextGenerationEU/PRTR. V.C. acknowledges Junta de Andalucía for financial support (POSTDOC_21_00694).File List: - Fig1b_KineticScansSAOEuDy.txt : Time (s, column 1), normalized intensity kinetic scans of empty sphere at 400 nm used to calculate Abs (column 2), SAO:Eu,Dy at 400 nm used to calculate Abs (column 3) and SAO:Eu,Dy at 524 nm (column 4). - Fig1c_PersLSpectrumSAOEuDy.txt : Wavelength (nm, column 1), normalized PersL intensity of SAO:Eu,Dy (column 2). - Fig1d_LumSpectrumSAOEuDy.txt : Wavelength (nm, column 1), normalized Lum intensity of SAO:Eu,Dy (column 2). - Fig2a_QYSAOEuDyChargingTime.txt : Charging time (s, column 1), LumQY of SAO:Eu,Dy (column 2), error of LumQY of SAO:Eu,Dy (column 3), PersLQY of SAO:Eu,Dy (column 4) and error of PersLQY of SAO:Eu,Dy (column 5). - Fig2b_ChargingKineticSAOEuDy.txt : Wavelength (nm, column 1), normalized Lum intensity of SAO:Eu,Dy (column 2). - Fig3c_SpectraZGOCr.txt : Wavelength (nm, column 1), normalized Lum intensity of ZGO:Cr (column 2), Wavelength (nm, column 3) and normalized PersL intensity of ZGO:Cr (column 4). - Fig3d_KineticScansZGOCr.txt : Time (s, column 1), normalized intensity kinetic scans of empty sphere at 270 nm used to calculate Abs (column 2), ZGO:Cr at 260 nm used to calculate Abs (column 3) and ZGO:Cr at 696 nm (column 4). - Fig3e_PersLESpectrumZGOCr.txt : Wavelength (nm, column 1) and normalized PersLE intensity of ZGO:Cr (column 2). - Fig4a_IntegratedPersLTemperature.txt : Time after excitation (s, column 1), normalized integrated intensity of ZGO:Cr at RT after excitation at 270 nm (column 2), normalized integrated intensity of ZGO:Cr after excitation at 270 nm with heating at 85 ºC (column 3), Time after excitation (s, column 4), normalized integrated intensity of ZGO:Cr at RT after excitation at 330 nm (column 5), normalized integrated intensity of ZGO:Cr after excitation at 330 nm with heating at 85 ºC (column 6), normalized integrated intensity of SAO:Eu,Dy at RT after excitation at 400 nm (column 7), normalized integrated intensity of SAO:Eu,Dy after excitation at 400 nm with heating at 85 ºC (column 8).Peer reviewe