Doxorubicin is an anthracycline chemotherapeutic produced by Streptomyces peucetius . It is an effective chemotherapeutic but has severe side effects and high toxicity, and the details of its biosynthetic pathway are largely unknown. Baumycin is a further decorated form of doxorubicin with an additional seven‐carbon acetal moiety attached to the anthracycline backbone of the molecule. In this work, we report our progress on the characterization of DnmZ, a Class D flavin monooxygenase involved in formation of the baumycin acetal. DnmZ is a N‐oxygenase that oxidizes an sugar‐linked exocyclic amine, triggering a nonenzymatic retro oxime‐aldol cleavage of the hexose. We aim to characterize the kinetics of the DnmZ‐catalyzed reaction between flavin and oxygen using stopped flow spectrophotometry. Results from this research can aid in the development of derivatized anthracyclines and the understanding of the class of enzymes known as the class D flavin monooxygenases. Our transient‐state experiments confirm that DnmZ catalyzes the formation of the C4a‐(hydro)peroxyflavin intermediate (C4a‐OOH). We have identified the appropriate wavelengths to monitor to observe C4a‐OOH formation (378nm) and elimination (450 and 480 nm). With this information we have quantitated flavin binding and determined that DnmZ’s binding affinity for flavin is approximately 13 μM, which compares well with other Class D monooxygenases. We are also investigating the effect of pH (pH 5.5 to 10.5) on C4a‐OOH formation and elimination. Our preliminary results suggest that increasing pH speeds up C4a‐OOH elimination, but has no effect on the intermediate’s formation. Support or Funding Information: California State University Program for Education & Research in BiotechnologyNIH SCORE 5SC3GM122652