Bacteriophages (phages) are viruses that infect bacteria. Some phages chemically modify their genomes to protect them from degradation by bacterial immune systems. We can detect phage genome modifications with mass spectrometry (MS) and high-performance liquid chromatography (HPLC) of phage nucleosides. This collection contains protocols for the entire process: phage growth and concentration, DNA extraction, and chemical analysis of phage nucleosides. We optimized these protocols using E. coli phage T4 and B. subtilis phage SPO1.