Heat shock response in Drosophila melanogaster is a complex process causing changes of the cellular environment in reaction to the heat shock induction. RNA transcription is dramatically changed during heat shock response and many regulatory factors are required. Chromatin insulators are found to be involved in the transcriptional regulation process, but much remains to be understood on how insulator function. Described herein are multiplexed selections and characterization of inhibitory RNA aptamers against several Drosophila insulator proteins using an efficient selection method, RAPID (RNA APtamer Isolation via Dual-cycles), and high-throughput sequencing. Background binding sequences are identified through a series of comprehensive analyses on the sequencing dataset. I have also examined the binding affinity of the top aptamer candidates and designed assays to map the binding elements. Aptamers generated here can be utilized as a very powerful approach to dissect the insulator function in vivo in the well-characterized heat shock system.