RNA extraction has been improved by integration of a variety of materials in the protocol, such as phenol, guanidine thiocyanate, and silica, according to the case-specific demands. However, few methods have been designed for high-throughput RNA preparation for large-scale transcriptome studies. In this study, we established a high‐throughput guanidinium thiocyanate and isopropyl alcohol based RNA extraction method (HighGI). HighGI is based on simple and phenol-free homemade buffers and the cost is substantially lower than a column-based commercial kit. We demonstrated that the quality and quantity of RNA extracted with HighGI were comparable to those extracted with a conventional phenol/chloroform-based method and a column-based commercial kit. HighGI retained small RNAs less than 200 bp, which are lost with a commercial column-based kit. We also demonstrated that HighGI is readily applicable to semi-automated RNA extraction. HighGI enables high‐throughput RNA extraction for large-scale RNA preparation with high yield and quality.