Phytobricks are standardized DNA parts for plants that can be assembled hierarchically into transcriptional units and, subsequently, into multigene constructs. Phytobricks each contain the sequences of one or more functional elements that comprise eukaryotic transcription units, with sequence features that enable them to be used interchangeably in one-step cloning reactions to facilitate combinatorial assembly. The simplicity and efficiency of this one-step reaction has enabled Phytobrick assembly to be miniaturized and automated on liquid handing platforms. In this method, we describe how to design and construct new Phytobricks as well as how to assemble them in both manual and nanoscale automated one-step reactions. Finally, we describe a high-throughput method for sequence verification of assembled plasmids.