Biopharmaceuticals produced in mammalian cell systems are in wide-spread use to treat and cure critical diseases. Chinese hamster ovary cells (CHO) still represent the major production host, but a number of novel cell lines were established in recent years. CEVEC’s Amniocyte Production (CAP®) cells are among these upcoming potential workhorses due to their ethically derived human background with authentic human glycosylation capacity combined with the ability to produce high titers of high-quality and even difficult to express proteins. CAP® cell lines expressing either secreted embryonic alkaline phosphatase (SEAP) or an IgG antibody were used in a novel high-content, high-throughput siRNA screening approach in complex chemically defined production medium in a micro-scale batch process in shaked microtiter plates. A custom library of 763 siRNAs against human kinases was used to identify potentially process-relevant kinases and critical cellular pathways by quantification of proliferation, viability, recombinant protein productivity and cell death (apoptosis). Kinases are among the key players in signal-transduction and represent highly attractive targets for cell line engineering approaches. A quarter of all siRNA mediated kinase knockdowns resulted in a significant modulation of at least one process-relevant parameter in SEAP producing CAP® cells. The strongest detrimental effects on proliferation were induced by knockdown of cell cycle kinases. Depletion of Polo-like kinase 1 (PLK1), Checkpoint kinase 1 (CHEK1) and Wee1-like protein kinase 1 (WEE1) reduced the viable cell concentration by more than 50 % in CAP®-SEAP cells. The same effect was verified in antibody producing CAP®-IgG cells. By applying PANTHER and INGENUITY® pathway analysis members of multiple growth factor signaling pathways and the PI3K-AKT-mTOR pathway (e.g. PIK3CA, PIK3R2, PIK3R3, PIK3C2B, and MAP3K2) were identified to be crucial for proliferation in CAP® cells. A third of all kinase knockdowns that led to reduced viable cell concentration had detrimental effects on recombinant protein production, showing the importance of high cell numbers for high-yielding batch processes. However, depletion of kinases like Endoplasmic-reticulum to nucleus signaling kinase 1 (ERN1), a member of the untranslated protein response (UPR) pathway, resulted solely in reduced recombinant protein yields without significant changes in proliferation. Anti-Serine-Threonine kinase 24 (STK24) and Anti-Death-associated protein kinase 3 (DAPK3) siRNA mediated knockdown showed elevated SEAP and IgG concentration in the screening. To answer the question, if overexpression of ERN1 could reverse the effects seen in the siRNA knockdown and lead to higher productivity, ERN1 was transiently expressed in CAP®-SEAP cells. This resulted in reduced proliferation and viability. Stable expression by CAP®-SEAP-ERN1 pools showed no beneficial effects on productivity, indicating that in contrast to CHO cells this kinase or the UPR pathway does not present a bottleneck in a CAP®-based recombinant protein process. The transient knockdown of STK24 improved IgG productivity in a five-day batch cultivation, whereas DAPK3 showed detrimental effects on both proliferation and productivity. Thus, STK24 presented a putative target for cell line engineering by stable knockdown or knockout. The study identified and verified kinases and pathways crucial for proliferation and productivity in CAP®-SEAP and CAP®-IgG cells that represent potential candidates for further cell line engineering approaches.