To determine optimal constrained tissue parameters and off-resonance sequence parameters for single-point macromolecular proton fraction (SP-MPF) mapping based on a comprehensive quantitative magnetization transfer (qMT) protocol in healthy and demyelinated living mice at 7T. Methods: Using 3D spoiled gradient echo-based sequences, a comprehensive qMT protocol is performed by sampling the Z-spectrum of mice brains, in vivo. Provided additional T 1 , B + 1 and B 0 maps allow for the estimation of qMT tissue parameters, among which three will be constrained, namely the longitudinal and transverse relaxation characteristics of the free pool (R 1,f T 2,f), the cross-relaxation rate (R) and the bound pool transverse relaxation time (T 2,r). Different sets of constrained parameters are investigated to reduce the bias between the SP-MPF and its reference based on the comprehensive protocol. Results: Based on a whole-brain histogram analysis about the constrained parameters, the optimal experimental parameters that minimize the global bias between reference and SP-MPF maps consist of a 600° and 6 kHz off-resonance irradiation pulse. Following a Bland-Altman analysis over regions of interest, optimal constrained parameters were R 1,f T 2,f = 0.0129, R = 26.5 s −1 , and T 2,r = 9.1 µs, yielding an overall MPF bias of 10 −4 (limits of agreement [−0.0068;0.0070]) and a relative variation of 0.64% ± 5.95% between the reference and the optimal single-point method across all mice. Conclusion: The necessity of estimating animal model-and field-dependent constrained parameters was demonstrated. The single-point MPF method can be reliably applied at 7T, as part of routine preclinical in vivo imaging protocol in mice.