Neuroinflammation has been implicated in amyotrophic lateral sclerosis (ALS) and can be visualized using translocator protein (TSPO) radioligands. To become a reliable pharmacodynamic biomarker for ALS multicenter trials, TSPO radioligands have some challenges to overcome. We aimed to investigate whether multicenter data pooling of different TSPO tracers (C-PBR28 and F-DPA714) is feasible, after validation of an established C-PBR28 PET pseudo reference analysis technique for F-DPA714. Seven ALS patients from Belgium (58.9 ± 6.7 y old, 5 men and 2 women), 8 healthy volunteers from Belgium (52.1 ± 15.2 y old, 3 men and 5 women), 7 ALS patients from the United States (53.4 ± 9.8 y old, 5 men and 2 women), and 7 healthy volunteers from the United States (54.6 ± 9.6 y old, 4 men and 3 women) from a previously published study underwent dynamic F-DPA714 (Leuven, Belgium) or C-PBR28 (Boston, Massachusetts) PET/MRI. For F-DPA714, maps of total volume of distribution (V) were compared with SUV ratio (SUVR) images from 40 to 60 min after injection (SUVR) calculated using the pseudo reference regions cerebellum, occipital cortex, and whole brain (WB) without ventricles. For C-PBR28, SUVR images from 60 to 90 min after injection using the WB without ventricles were calculated. In line with previous studies, increased F-DPA714 uptake (17.0% ± 5.6%) in primary motor cortices was observed in ALS subjects, as measured by both V and SUVR approaches. The highest sensitivity was found for SUVR calculated using the WB without ventricles (average cluster, 21.6% ± 0.1%). F-DPA714 V ratio was highly correlated with the SUVR ( > 0.8, < 0.001). A similar pattern of increased uptake (average cluster, 20.5% ± 0.5%) in the primary motor cortices was observed in ALS subjects for C-PBR28 SUVR calculated using the WB without ventricles. Analysis of the F-DPA714 and C-PBR28 data together resulted in a more extensive pattern of significantly increased glial activation bilaterally in the primary motor cortices. The same pseudo reference region analysis technique for C-PBR28 PET can be extended toward F-DPA714 PET. Therefore, in ALS, standardized analysis across these 2 tracers enables pooling of TSPO PET data across multiple centers and increases the power of TSPO as a biomarker for future therapeutic trials.