Diagnosis of primary glial malignancies (GLI-M) in individuals presenting with Intracranial Space Occupying Lesions (ICSOL) is based on histopathological evaluation (HPE) of tissue obtained by surgical resection or biopsy with attendant resource implications and risks. Approximately 70% of ICSOLs have non-malignant etiology and distinction from malignant lesions rests largely on HPE. Furthermore, GLI-M must be differentiated from metastatic lesions to the brain arising from solid tumors in other organs. We describe a blood-based test which detects GLI-M with high sensitivity and differentiates it from benign brain tumours (BBT) and brain metastases. The test is based on fluorescent multiplexed immunocytochemical (ICC) profiling for identification of Circulating Glial Cells (CGCs) and Circulating (Epithelial) Tumor Cells (CTCs) enriched from peripheral blood. The performance characteristics of the test were established in analytical validation as well as clinical studies. In an initial case-control study with discrete training (n = 31 BBT and n = 101 GLI-M) and test (n = 13 BBT and n = 44 GLI-M) sets, our platform showed 100% sensitivity and 100% specificity in identifying and differentiating GLI-M from BBT in the test set. In a prospective study of 68 individuals presenting with ICSOL, the test had 100% sensitivity and 100% specificity for identifying and differentiating GLI-M (n = 56) and BBT (n = 12). Finally, in a subset analysis of 586 samples, the test accurately identified samples from GLI-M (n = 40), BBT (n = 22), epithelial malignancies (EPI-M, n = 24) and healthy individuals (n = 500), with no false positive or false negative findings. The performance characteristics of this blood-based platform support its utility in clinical practice for diagnostic triaging of individuals presenting with ICSOL and facilitating more effective diagnosis compared to standard of care.