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Although small extracellular vesicles (sEVs) have promising features as an emerging therapeutic format for a broad spectrum of applications, for example, blood–brain‐barrier permeability, low immunogenicity, and targeted delivery, economic manufacturability will be a crucial factor for the therapeutic applicability of sEVs. In the past, bioprocess optimization and cell line engineering improved titers of classical biologics multifold. We therefore performed a design of experiments (DoE) screening to identify beneficial bioprocess conditions for sEV production in HEK293F suspension cells. Short‐term hyperthermia at 40°C elevated volumetric productivity 5.4‐fold while sEVs displayed improved exosomal characteristics and cells retained >90% viability. Investigating the effects of hyperthermia via transcriptomics and proteomics analyses, an expectable, cellular heat‐shock response was found together with an upregulation of many exosome biogenesis and vesicle trafficking related molecules, which could cause the productivity boost in tandem with heat shock proteins (HSPs), like HSP90 and HSC70. Because of these findings, a selection of 44 genes associated with exosome biogenesis, vesicle secretion machinery, or heat‐shock response was screened for their influence on sEV production. Overexpression of six genes, CHMP1A, CHMP3, CHMP5, VPS28, CD82, and EZR, significantly increased both sEV secretion and titer, making them suitable targets for cell line engineering. [ABSTRACT FROM AUTHOR]