The success of messenger RNA (mRNA)-based vaccines against SARS-CoV-2 during the COVID-19 pandemic has driven significant advancements in mRNA-based therapeutics. Key innovations include the use of modified nucleosides and ionizable lipid nanoparticles. Modified mRNA is produced through in vitro transcription, typically employing phage T7 RNA polymerase along with a DNA template and ribonucleoside triphosphates. Chemical modifications at the 5-position of pyrimidines, (cytosine and uridine) are generally well tolerated by T7 RNA polymerase and can reduce innate immune activation, though their effects on translation vary considerably. Therefore, the nature and codon-positions of these modifications, as well as the mRNA sequence, should be considered when designing mRNA therapeutics. This chapter details the production, purification, and evaluation of translation in human cells for modified mRNA containing 5-methyluridine, 5-methoxyuridine, 5-hydroxymethyluridine, pseudouridine, N1-methylpseudouridine, or N1-ethylpseudouridine.