Laboratory automation impacts nearly all aspects of high throughput RNAi screens. It is particularly relevant when considering library format and storage, and also when planning high throughput transfection protocols. In situations where libraries are stored as screening copies that are used for just-in-time dispensing, automation can be utilized as a tool to accommodate diverse assay and cell types and can enable forward or reverse transfections into a variety of different microplates. Automation has the ability to increase the feasibility and decrease consumable costs for assays that require a large number of replicates. It is also an important tool when considering more complex assays, such as those that utilize non-standard plate types or electroporation, as automation increases reliability and can improve assay performance. This chapter highlights important considerations for library formatting and ways in which laboratory automation can be implemented to facilitate RNAi high throughput screening.