Scandium-44 (t_1/2 = 3.9 h) is a relatively new radioisotope of potential interest for use in clinical positron emission tomography (PET). Here in, we report, for the first time, the room-temperature radiolabeling of proteins with ⁴⁴Sc for in vivo PET imaging. For this purpose, the Fab fragment of Cetuximab, a monoclonal antibody that binds with high affinity to epidermal growth factor receptor (EGFR), was generated and conjugated with N-[(R)-2-amino-3-(paraisothiocyanato- phenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine- N,N,N′,N″,N″-pentaacetic acid (CHX-A″-DTPA). The high purity of Cetuximab-Fab was confirmed by SDS-PAGE and mass spectrometry. The potential of the bioconjugate for PET imaging of EGFR expression in human glioblastoma (U87MG) tumor-bearing mice was investigated after ⁴⁴Sc labeling. PET imaging revealed rapid tumor uptake (maximum uptake of ~ 12% ID/g at 4 h postinjection) of ⁴⁴Sc−CHX-A″-DTPA− Cetuximab-Fab with excellent tumor-to-background ratio, which might allow for same day PET imaging in future clinical studies. Immunofluorescence staining was conducted to correlate tracer uptake in the tumor and normal tissues with EGFR expression. This successful strategy for immunoPET imaging of EGFR expression using ⁴⁴Sc−CHX-A″-DTPA−Cetuximab-Fab can make clinically translatable advances to select the right population of patients for EGFR-targeted therapy and also to monitor the therapeutic efficacy of anti-EGFR treatments.