The methylation status of the MGMT promoter represents the valuable prognostic and predictive marker in glioblastoma (GBM) patients undergoing treatment with alkylating agents such as Temozolomide. Although Formalin-Fixed and Paraffin-Embedded Tissue (FFPE) signifies the most commonly used source for tissue-based molecular testing, its use in Methylation-Specific Polymerase Chain Reaction (MSP) analysis manifests certain limitations due to low DNA integrity. Our study aimed to identify the optimal MGMT promoter MSP reaction conditions concerning the utilization of bisulfite-converted FFPE-derived template DNA. Several optimizing reactions were conducted and subjected to ImageJ software analysis. As a result, 4U of HotStarTaq and 125 ng of template DNA were specified as necessary for successful MSP reactions. The confirmation of optimization success was obtained through comparison of semi-quantitative values of DNA methylation levels between reference Fresh Frozen tissue and corresponding FFPE sample obtained from the same GBM patient.