Cytokinins (CKs) are phytohormones produced by plants and other soil life. including bacteria, fungi, insects, and earthworms. These organisms can release CKs to the soil, which may have positive implications for soil health and plant growth. However, no studies have examined phytohormones as soil health indicators. In custom-designed rhizo-pots that separated rhizosphere and bulk soils, the cover crops tillage radish and cereal rye were used to manipulate soil health parameters: soil pH, soil organic matter, soil active carbon, soil microbial community diversity, and extracellular enzyme activities involved in C, N and P cycling. Data were compared to impacts of cover crops on CKs that were purified from the complex soil and measured with HPLC-HRMS/MS. From soil we detected free base-CKs (trans-zeatin (tZ), isopentenyladenine (iP)), riboside-CKs (RB-CKs), cis-zeatin riboside (cZR), isopentenyladenosine (iPR) and four methylthiolated CKs: 2-methylthio-zeatin (2MeSZ), 2-methylthio-zeatin ribosides (2MeSZR), 2-methylthio-isopentenyladenine (2MeSiP), and 2-methylthio-isopentenyladenine riboside (2MeSiPR). These CK levels were significantly enhanced in cover cropped soil compared to uncultivated soil, and reflect a positive relationship between soil CK profiles and other soil health parameters - notably, between total CK and active C levels and soil microbial community diversity. This is the first detailed soil CK analysis and assessment of its potential use as a novel, reliable, short-term soil health parameter. The increased CK concentrations in cover cropped soils likely reflects the activity levels of soil life (plants, microbes, animals) and provides a rationale to use CKs as tools to evaluate soil health as influenced by agricultural management strategies.