Background: PET with O-(2-F-18-fluoroethyl)-L-tyrosine (F-18-FET) has reached increasing clinical significance for patients with brain neoplasms. For quantification of standard PET-derived parameters such as the tumor-to-background ratio, the background activity is assessed using a region of interest (ROI) or volume of interest (VOI) in unaffected brain tissue. However, there is no standardized approach regarding the assessment of the background reference. Therefore, we evaluated the intra-and inter-reader variability of commonly applied approaches for clinical F-18-FET PET reading. The background activity of 20 F-18-FET PET scans was independently evaluated by 6 readers using a (i) simple 2D-ROI, (ii) spherical VOI with 3.0 cm diameter, and (iii) VOI consisting of crescent-shaped ROIs;each in the contralateral, non-affected hemisphere including white and gray matter in line with the European Association of Nuclear Medicine (EANM) and German guidelines. To assess intra-reader variability, each scan was evaluated 10 times by each reader. The coefficient of variation (CoV) was assessed for determination of intra-and inter-reader variability. In a second step, the best method was refined by instructions for a guided background activity assessment and validated by 10 further scans. Results: Compared to the other approaches, the crescent-shaped VOIs revealed most stable results with the lowest intra-reader variabilities (median CoV 1.52%, spherical VOI 4.20%, 2D-ROI 3.69%;p < 0.001) and inter-reader variabilities (median CoV 2.14%, spherical VOI 4.02%, 2D-ROI 3.83%;p = 0.001). Using the guided background assessment, both intra-reader variabilities (median CoV 1.10%) and inter-reader variabilities (median CoV 1.19%) could be reduced even more. Conclusions: The commonly applied methods for background activity assessment show different variability which might hamper F-18-FET PET quantification and comparability in multicenter settings. The proposed background activity assessment using a (guided) crescent-shaped VOI allows minimization of both intra-and inter-reader variability and might facilitate comprehensive methodological standardization of amino acid PET which is of interest in the light of the anticipated EANM technical guidelines.