Diplonemids are heterotrophic marine flagellates that have attracted attention and interest due to their eccentric mitochondrial genome and gene expression. This has led to the development and optimization of experimental procedures aimed at better understanding of the unusual mitochondrial processes that occur in this group. Here we present a collection of protocols established in the diplonemid type species Paradiplonema papillatum, which range from DNA preparation for direct long-read sequencing of both mitochondrial and nuclear DNA, to isolation of mitochondria and separation of mitochondrial ribosomes, to assays for assessing metabolic functions through measurement of respiration, mitochondrial membrane potential, and peroxide-scavenging activities, as well as tracking metabolic routes via stable-isotope labeling of metabolic intermediates.