Aptamers are made up of DNA or RNA form a 3D structure that binds to specific targets including ions , small molecules , proteins , and microorganisms . Aptamers can be used for both therapeutics and diagnostics with several clinical trials being currently performed. There are currently diagnostic tests on the market used for diagnosing viral diseases like COVID-19 using aptamers . Although several research groups have started making aptamers for other diagnostic purposes there are few that have been marketed on a wider scale. In the current study, Thiazole Orange (TO) was used as an intercalating dye with specific aptamers that bind to cortisol to determine its concentration in saliva. TO is a forced intercalation probe (FIT probe or light up probe) that can bind oligonucleotides (DNA or RNA) which can bind as a monomer or a dimer and intercalate into their minor grooves . The fluorescent intensity of TO increases when it is bound and reduces when unbound in solution. TO has a specific affinity with a higher quantum yield when bound to double stranded DNA or to DNA containing polyA or polyG . It also tends to bind to specific aptamer structures like the G hairpin . The TO probe can be used to distinctly measure the concentration of a biomarker e.g. vitamins and hormones by molecular displacement. Upon binding between aptamer and hormone, the bound TO is displaced from the nucleotide resulting in an decrease in fluorescent intensity. In the current work, we demonstrate an aptamer detection system that can be used to detect different hormones and vitamins in saliva samples in a 96-well plate format. We performed experiments both in buffer and artificial saliva matrix and propose this as a novel way and inexpensive to detect cortisol in saliva.