Introduction: Sarcomas are mesenchymal tumors often associated with therapeutical resistance and recurrence. Immunotoxins (ITs) represent a promising strategy, joining the specificity of carriers to the cytotoxicity of plant toxins (Ribosome-Inactivating Protein, RIP). Three-dimensional models represent a ground-breaking advance in cancer therapy, especially for predictive high-throughput drug screening. Our study aimed to comparatively evaluate the IT efficacy to TfR1-, EGFR1- and Her2-expressing sarcoma cells, in 2D (adherent cells) and 3D (spheroids) models. Methods Three different ITs were obtained by chemical conjugation of the RIP Saporin to Transferrin (Tf-IT) and of the RIP Ocymoidine to α-EGFR1 (α-EGFR1-IT) and to α-Her2 (α-Her2-IT). The IT efficacy was evaluated on rhabdomyosarcoma (RD18) and osteosarcoma (U2OS), in dose–response viability experiments (MTS colorimetric assay for the 2D model and ATP luminometric assay for the 3D model) and using a Caspase 3/7 activation assay. Results: In the RD18 and U2OS 2D and 3D models, the RIP cytotoxicity was significantly enhanced after chemical conjugation to carriers. The ITs strongly reduced cell viability, showing EC₅₀ values 1.3-2.3 logs lower than the corresponding unconjugated RIP and carrier. Despite the increased complexity and penetration difficulty characterizing 3D models, ITs were also effective in 3D models, showing similar EC₅₀ compared to 2D ones. All the ITs were able to strongly activate Caspase 3/7. Conclusions: Our data showed the strong efficacy of all tested ITs. The strategy of targeting different antigens on tumour-cells' surfaces may also allow us to kill clones expressing mutated target antigens. RIPs trigger different cell-death mechanisms, avoiding the selection of drug-resistant neoplastic clones. Thus, our results support the potential use of ITs as a therapeutic strategy in the treatment of sarcomas. Further experiments will be necessary to evaluate "in vivo" the tolerable doses of ITs and to compare their efficacy with the drugs approved by the FDA for sarcoma therapy.