Epigenetic regulation affects diverse diseases, and high throughput screening for histone methyltransferase (HMT) inhibitors is an area of intense activity. HMTs produce many different methylated products, and assay methods that detect S-adenosylhomocysteine (SAH) – the invariant product of S-adenosylmethionine (SAM)-dependent methylation reactions - are therefore advantageous over methods that detect specific methylation events. However, direct detection of SAH requires a reagent capable of discriminating between SAH and SAM, which differ by a single methyl group. There is a significant sensitivity challenge as well because HMTs are slow enzymes and use low levels of SAM. Currently there are no SAH assays with sufficient selectivity and sensitivity to allow universal detection HMTs under typical screening conditions, and researchers are resorting to alternative approaches that require intensive assay development and/or are less amenable to HTS. To overcome this technical gap, we leveraged a naturally occurring SAH-sensing RNA aptamer, or “riboswitch”, that binds SAH with nanomolar affinity and exquisite selectivity. We showed that binding of SAH to the riboswitch can be transduced into fluorescence polarization (FP) and time resolved Förster resonance energy transfer (TR-FRET) signals. Surprisingly, we found that splitting the riboswitch into two halves, such that SAH binding induces assembly of a trimeric complex, improved the sensitivity, selectivity and stability of the signaling. We used the split aptamer assays to detect SAH produced by purified HMTs at low nanomolar levels - several-fold below the sensitivity limit for current assays – using diverse acceptor substrates ranging from peptides to intact nucleosomes. These riboswitch-based SAH sensors provide the basis for sensitive, robust, universal detection of HMTs in an HTS-compatible format.