Purpose: Recent studies have shown rapid accumulation of nanobodies (NBs) in tumors and fast clearance of the unbound fraction, making NBs exceptional tracers for cancer imaging. In this study, we investigate the combination of in vitro imaging of tumor spheroids, in vivo dual-isotope single-photon emission computed tomography (SPECT), and ex vivo autoradiographic analysis of tumors to efficiently, and with few mice, assess the tumor uptake and distribution of different NBs. Procedures: The irrelevant NB R2 (16 kDa) and the EGFR-targeted NBs 7D12 (16 kDa) and 7D12-R2 (32 kDa) were investigated. Confocal microscopy was used to study the penetration of the NBs into A431 tumor spheroids over time, using the anti-EGFR monoclonal antibody (mAb) cetuximab (150 kDa) as a reference. Dual-isotope [ 111 In]DOTA-NB/[ 177 Lu]DOTA-NB SPECT was used for longitudinal imaging of multiple tracers in the same animal bearing A431 tumor xenografts. Tumor sections were analyzed using autoradiography. Results: No binding of the irrelevant NB was observed in spheroids, whereas for the specific tracers an increase in the spheroid’s covered area was observed over time. The NB 7D12 saturated the spheroid earlier than the larger, 7D12-R2. Even slower penetration was observed for the large mAb. In vivo, the tumor uptake of 7D12 was 19-fold higher than R2 after co-injection in the same animal, and 2.5-fold higher than 7D12-R2 when co-injected. 7D12-R2 was mainly localized at the rim of tumors, while 7D12 was found to be more evenly distributed. Conclusions: This study demonstrates that the combination of imaging of tumor spheroids, dual-isotope SPECT, and autoradiography of tumors is effective in comparing tumor uptake and distribution of different NBs. Results were in agreement with published data, highlighting the value of monomeric NBs for tumor imaging, and re-enforcing the value of these techniques to accurately assess the most optimal format for tumor imaging. This combination of techniques requires a lower number of animals to obtain significant data and can accelerate the design of novel tracers.