<p>Synthetic biology reprograms organisms to perform non-native functions for beneficial reasons. An important practice in synthetic biology is the ability to edit DNA to change a base pair, disrupt a gene, or insert a new DNA sequence. DNA edits are commonly made with the help of homologous recombination, which inserts new DNA flanked by sequences homologous to the target region. To increase homologous recombination efficiency, a double stranded break is needed in the middle of the target sequence. Common methods to induce double stranded breaks use nucleases, enzymes that cleave ribonucleotides (DNA and RNA). The most common nucleases are restriction enzymes, which recognize a short, fixed, palindromic DNA sequence (4-8 base pairs). Because of the short and fixed nature of the recognition sites, restriction enzymes do not make good candidates to edit large chromosomal DNA. Alternatively, scientists have turned to programmable endonucleases which recognize user-defined DNA sequences, often times much larger than the recognition sites of restriction enzymes (15-25 base pairs). Programmable endonucleases such as CRISPR-based systems and prokaryotic Argonautes are found throughout the prokaryotic kingdom and may differ significantly in activity and specificity. To compare activity levels among endonuclease enzymes, activity assays are needed. These assays must clearly delineate dynamic activity levels of different endonucleases and work with a wide variety of enzymes. Ideally, the activity assay will also function as a positive selection screen, allowing modifications to the enzymes via directed evolution. Here, we develop an <i>in vivo</i> assay for programmable endonuclease activity that can also serve as a positive selection screen using two plasmids, a lethal plasmid to cause cell death and a rescue plasmid to rescue cell growth. The lethal plasmid houses the homing endonuclease, I-SceI, which causes a deadly double-stranded break at an 18 base pair sequence inserted into an engineered <i>E. coli</i> genome. The rescue plasmid encodes for a chosen endonuclease designed to target and cleave the lethal plasmid, thereby preventing cell death. With this, cell growth is directly linked to programmable endonuclease activity. Three endonucleases were tested, SpCas9, eSpCas9, and xCas9, displaying recovered growth of 49.3%, 26.1%, and 16.4% respectively. These values translate to kinetic enzymatic activity and are congruent with current literature findings as reported values find WT-SpCas9 to have the fastest kinetics cleaving around 95% of substrate within 15 seconds, followed closely by eSpCas9 cleaving 75% of substrate within 15 seconds and finally trailed by xCas9 cleaving 20% of substrate in about 30 seconds. The differences between each endonuclease’s activity is exacerbated in our <i>in vivo</i> system when compared to similar <i>in vitro</i> methods with much lower resolution. Therefore, slight differences in activity between endonucleases within the first few minutes in an <i>in vitro</i> assay may be a few percentages different whereas in our <i>in vivo</i> assay, these differences in activity result in a more amplified signal. With the ability to display the dynamic response of enzymes, this assay can be used to compare activity levels between endonucleases, give insight into their kinetics, and serve as a positive selection screen for use in directed evolution applications. </p>