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Nickel-based superalloys are the material of choice for use in the hottest regions of gas turbine engines due to their inimitable mechanical properties, in particular, high temperature strength. As we become increasingly aware of the environmental consequences of air travel, manufacturers are striving to improve engine efficiency. This may be achieved by increasing the operating temperature of the engine, however, the limiting factor to achieve this is the material used. In order to successfully design new alloys for use in such high temperature applications, a full understanding of the strengthening mechanisms is required. If this were possible, alloy properties such as composition and microstructure could be optimized to the specific mechanisms known to be occurring. However, this is a complex task, as many underlying mechanisms contribute to the superior high temperature strength of these materials, and the extent of each individual mechanism is contentious. The aim of this work was to gain a full understanding of the effect of microstructure on the yield strength of polycrystalline Ni-based superalloys, and the effect on and extent of each of the underlying strengthening mechanisms. To this end, model polycrystalline Ni-based superalloys were studied, based on the quinary Ni-Cr-Al-Ti-Mo system. The series contained 6 alloys with varying Mo content from 0 to 5 at.%, and, as typical of polycrystalline Ni-based superalloys, all had microstructures containing ordered γ' precipitates within the disordered γ matrix phase. These alloys were fully characterized using electron microscopy techniques in addition to neutron diffraction, to determine experimentally properties such as phase composition, particle size distribution, γ' volume fraction and grain size. Results were found to differ to commonly accepted wisdom in the literature. Accepted models from the literature for each of the underlying strengthening mechanisms were used to predict the contribution of each mechanism to the overall yield strength of the alloys in this model series. These results were compared to experimental data to determine the efficacy of the models. Microstructural and compositional data from the literature for a number of commercial polycrystalline alloys were also used as input for the models to determine their effectiveness at this more complex level. The results obtained indicate that our currently accepted understanding of yield strength modeling in Ni-based superalloys is insufficient and does not successfully incorporate all of the strengthening mechanisms that are occurring concurrently in these materials. To gain further understanding of the deformation processes that give rise to the overall yield strength of these materials, in situ neutron diffraction experiments were carried out to quantify the degree of load partitioning between grain orientations and between the γ and γ' phases. It was concluded that both intergranular and interphase load partitioning occur during loading, amid complex interaction between phase strength and coherency.