Novel and simple spectrophotometric and distance based procedures for thiols (L-cysteine, N-acetylcysteine, and glutathione) determination in biological fluids and pharmaceuticals have been proposed based on their inhibitory action on the oxidation of catechol in the presence of Agaricus bisporus crude extract (ABE). The influence of L-glycine, L-alanine, L-proline, L-methionine, L-cystine, ascorbic acid, uric acid, and bilirubin on the thiol determination has been investigated. Uric acid, bilirubin, L-cystine (oxidized thiol), and L-amino acids do not interfere with the determination. The interference of ascorbic acid up to 350 mg/L is eliminated by using Cucumis sativus crude extract (CSE) with ascorbate oxidase activity. Distance based microfluidic paper-based analytical device (DμPAD) has been developed using origami paper device approach and ABE-catechol-3-methyl-2-benzothiazolinone hydrazone (MBTH) system. DμPAD has the 3D structure of three layers: CSE microzone layer, ABE microzone layer, and chemometer layer (catechol + MBTH). This structure allows sequential sample treatment (ascorbic acid oxidation by CSE) and the following introduction of treated sample to ABE and its substrate to perform ABE inhibition. Separate loading of ABE and its substrate allows preventing their interaction prior to sample loading. DμPAD thiol determination is performed by measuring the length of uncolored flow channel, which allows very simple thiol determination and enables complete integration of analytical procedure steps: sample treatment and enzymatic determination with visual signal output. The analytical ranges are (0.3-2.5)∙10