Lentiviral vectors (LVV) represent an important tool for cell and gene therapy applications. Most of the current LVV production methods use the HEK293T cell line as it is easily transfected and supports high-level expression of viral proteins. However, the inability to achieve high cell densities (i.e. >10 × 106 cells/mL) with HEK293T stable cell lines in a fed batch process have resulted in poor upstream yields. Optimisation of cell culture conditions is needed to improve upstream yields, which can be expedited by high-throughput screening. This thesis describes the use of Liquid Chromatography-Mass Spectrometry (LC-MS) tools and high-throughput screening to develop a novel feed for improving the growth profile of GSK’s 277 HEK293T LVV stable producer cell line, thus enabling high titre lentiviral vector production. Firstly, LC-MS analysis was used to generate a profile of metabolite changes during stable cell line cultivation in 2 L stirred tank reactors (STR). A total of 50 metabolites were identified to be depleting with time, and thus possible targets for developing a bespoke feed. Thereafter, the 24 deep square well (24-DSW) microwell platform was used to develop a scale-down mimic of GSK’s established stable suspension LVV production process model at 2 L STR scale. Matched mixing time was found to be an effective basis for scale-translation between the STR and microwells. The growth kinetics and LVV productivity profile in the microwell were reproducible and comparable to the 2 L STR process model as defined by the maximum VCD prior to stationary phase (~6 × 106 cells/mL) and infectious titre at harvest (~2 × 107 TU/mL). A design of experiments (DoE) approach, facilitated by the 24-DSW high-throughput model, was then used to screen and optimise feeds based upon the depleting metabolites identified, and thus develop a novel feed for optimised HEK293T stable cell line culture. Using the novel feed, the period of exponential growth was increased by up to 24 hours which enabled induction at higher cell densities. This resulted in a 2-fold increase in infectious titre for the 277 stable cell line compared to the platform process control. Therefore, the 24-DSW model together with LC-MS analysis provides an important tool for rapid, high-throughput optimization of the LVV production process.