<p>Glioblastomas (GBMs) functionally integrate into diverse neuronal circuits within the central nervous system, which can promote tumor progression and affect neurons via neuron-to-glioma synapses. It remains challenging to identify and manipulate tumor-innervating neurons, which may remain localized or widely distributed throughout the brain. Building on GBM organoids (GBOs) derived from patient-resected surgical tissue, we present here detailed procedures for assessing interactions between tumors and neurons. We first discuss retrograde trans-monosynaptic tracing approaches to study the neuron–tumor connectome by using a rabies viral system in ex vivo human tissue and in xenogenic animal models. As a complementary approach, we then describe the use of anterograde transsynaptic tracing using herpes simplex virus in vivo and ex vivo to assess brain region-specific connectivity in GBMs. In addition, to facilitate the adaptability of these tracing methodologies in diverse systems, we provide procedures for the viral transduction into GBOs, the generation of assembloids comprising GBOs and human induced pluripotent stem cell-derived cortical organoids and the establishment of air–liquid interface cultures from surgical human brain tissue. Together, these techniques permit the flexible characterization and manipulation of tumor–neural circuits and can be easily adapted to other cancers with nervous system involvement. After the generation of GBOs and/or cortical organoids, transsynaptic tracing requires 12–35 d to complete ex vivo or in vivo. The procedure is suitable for users with expertise in human cell and organoid culture, viral production and transduction, rodent surgery and microscopy.</p>