Glioma is the most common primary intracranial tumor, with an overall median survival of 15 months from the time of diagnosis. The current gold standard for diagnosis is tumor tissue analysis for the detection of known mutations. Tissue sampling is done intraoperatively and current testing protocols utilize multiple PCR or sequencing-based assays. Moreover, the median time to diagnosis, from the time of tissue testing, is three weeks. This can lead to delays in diagnosis, patient stratification, treatment initiation, and patient selection for potential clinical trial enrollment. Rapid and cost-effective molecular subtyping of gliomas is therefore crucial to overcome these challenges. To achieve this, we propose a novel multiplexed digital PCR platform (QIAcuity Digital PCR System) for simultaneous detection of hallmark DNA- and RNA-based alterations specific to gliomas: IDH.R132H, EGFRvIII, and TERT.C228T/C250T. Key parameters were optimized, including, combined DNA/RNA isolation protocol, reverse transcription, probe dyes, thermocycling conditions, and use of additives. Preliminary testing in tumor tissue and cerebrospinal fluid (CSF) has yielded promising results, enabling sensitive and specific diagnosis of low-grade (astrocytoma, oligodendroglioma) and high-grade (glioblastoma) gliomas. Future efforts are focused on validating the assay performance in discovery and blinded validation cohorts comprising tumor tissue and matched CSF and plasma samples.