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<i>Spongospora subterranea</i> is a soil-borne plant pathogen responsible for the economically significant root and powdery scab diseases of potato. However, the obligate biotrophic nature of <i>S. subterranea</i> has made the detailed study of the pathogen problematic. Here, we first compared the benefits of sporosori partial purification utilizing Ludox^® gradient centrifugation. We then undertook optimization efforts for protein isolation comparing the use of a urea buffer followed by single-pot solid-phase-enhanced sample preparation (SP3) and a sodium dodecyl sulphate (SDS) buffer followed by suspension-trapping (S-Trap). Label-free, quantitative proteomics was then used to evaluate the efficiency of the sporosori purification and the protein preparation methods. The purification protocol produced a highly purified suspension of <i>S. subterranea</i> sporosori without affecting the viability of the spores. The results indicated that the use of a combination of SDS and S-Trap for sample clean-up and digestion obtained a significantly higher number of identified proteins compared to using urea and SP3, with 218 and 652 proteins identified using the SP3 and S-Trap methods, respectively. The analysis of proteins by mass spectrometry showed that the number of identified proteins increased by approximately 40% after the purification of spores by Ludox^®. These results suggested a potential use of the described spore purification and protein preparation methods for the proteomics study of obligate biotrophic pathogens such as <i>S. subterranea</i>.