Complete understanding of cellular conditions and monitoring of disease states rely on the quantitative and parallel measurement of cellular molecules. Nucleic acids can be amplified by PCR and detected in a high throughput manner by microarrays. Similarly, using nucleic acids as small molecule sensors, multiple small analytes can be detected in a parallel fashion. Natural riboswitches are one type of small molecule sensing RNAs that undergo conformational changes upon metabolite sensing and thereby regulate gene expression. In this study, a riboswitch was engineered with a molecular barcode (MB) label to detect small molecules by various approaches. MBs are a set of unique nucleotide sequences that serve as unique identifiers of a specific nucleic acid species from a mixed population. The incorporation of MB labels into the designed riboswitches allows multiple small molecules being quantified simultaneously using well established nucleic acid quantification technologies such as multiplexed PCR and oligonucleotide microarrays. Using nucleic acids as molecular recognition elements will also complement current array-based small-molecule detection techniques which may be limited by antibody availability and stability. As a proof of principle study, a theophylline MB-riboswitch was designed and used to quantify theophylline concentrations in solution. We found that the clinically relevant theophylline concentration (10-50 µM) was within the dynamic range of our technique (0.2 µM to 2 mM). Measurement of the thermodynamics and kinetics of the system produced conditions with the highest signal-to-noise ratio. Theophylline detection was also achieved by amplification of theophylline-bound riboswitches by real time-PCR, which significantly lowered the detection limit for the analyte.