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High-throughput light scattering instruments are widely used in screening of biopharmaceutical formulations and can be easily incorporated into processes by utilizing multi-well plate formats. High-throughput plate readers are helpful tools to assess the aggregation tendency and colloidal stability of biological drug candidates based on the diffusion self-interaction parameter (<i>k_D</i>). However, plate readers evoke issues about the precision and variability of determined data. In this article, we report about the statistical evaluation of intra- and inter-plate variability (384-well plates) for the <i>k_D</i> analysis of protein and peptide solutions. ANOVA revealed no significant differences between the runs. In conclusion, the reliability and precision of <i>k_D</i> was dependent on the plate position of the sample replicates and <i>k_D</i> value. Positive <i>k_D</i> values (57.0 mL/g, coefficients of variation (<i>CV</i>) 8.9%) showed a lower variability compared to negative <i>k_D</i> values (−14.8 mL/g, <i>CV</i> 13.4%). The variability of <i>k_D</i> was not reduced using more data points (120 vs. 30). A <i>k_D</i> analysis exclusively based on center wells showed a lower <i>CV</i> (<2%) compared to edge wells (5–12%) or a combination of edge and center wells (2–5%). We present plate designs for <i>k_D</i> analysis within the early formulation development, screening up to 20 formulations consuming less than 50 mg of active pharmaceutical ingredient (API).