This digital resource is a detailed workflow protocol for conducting one-color gene expression microarray experiments. One-color microarray analysis utilizes a single fluorescent label (e.g., Cy3) per sample, with each sample hybridized to a separate array. This contrasts with two-color arrays, where two samples are co-hybridized on a single array. The protocol outlines the entire process from RNA extraction to data analysis. The workflow typically includes steps such as total RNA extraction, quality assessment (e.g., using NanoDrop or Bioanalyzer), sample labeling with a single fluorescent dye (e.g., Cy3-UTP), cRNA purification and quantification, microarray hybridization design, blocking, hybridization, washing, and scanning. It emphasizes critical quality control points throughout the process to ensure reliable data. One-color microarrays are widely used for differential gene expression studies, treatment outcome evaluations, biomarker discovery, and time-course studies. A key advantage is the ease of experimental design, especially when the number of samples is large or unknown, as no reference sample is strictly required for comparison on the same array. This allows for greater flexibility in experimental setup and can be more cost-effective for certain study designs. This protocol is invaluable for researchers in genomics, molecular biology, and systems biology who are performing gene expression profiling. It provides a standardized approach to ensure high reproducibility and sensitivity in measuring gene expression levels, making it a foundational document for laboratories utilizing one-color microarray technology.