Patients with new-onset malignant spinal lesions often have an urgent need for local spine intervention and systemic therapy. For optimal management, it is crucial to diagnose the underlying disease as quickly and reliably as possible. The aim of our current study was to determine the feasibility, sensitivity, specificity, and diagnostic certainty of complementary cytological evaluation of spinal lesions suspected of malignancy. In 44 patients, we performed histopathological biopsies and in parallel cytologic preparations from the malignant site. Cytological smears were prepared and stained for May-Grunwald and Giemsa. Bone biopsies were histopathologically analyzed according to the existing standard-of-care practices. In 42 of 44 cases (95%), a cytological sample was successfully obtained. In 40 cases (95.2%, Cohen’s kappa: 0.77), the cytological diagnosis agreed with the histological diagnosis regarding the identification of a malignant lesion. This resulted in a sensitivity of 97% and a specificity of 80% as well as a diagnostic safety of 95%. Cytological analysis in the context of spinal surgery proved sufficient to establish a diagnosis of malignancy or its exclusion, expanding the existing diagnostic spectrum. Furthermore, implementation of this process as a routine clinical diagnostic might shorten the time to diagnosis and improve the treatment of this vulnerable patient group.