In the first study, a simple, cost-effective, instrumental free and user-friendly analytical method based on the combination of paper-based headspace extraction and digital image analysis was developed for the sensitive detection of cyanide in water and wastewater samples. The method relied on chloramine-T/pyridine-barbituric acid reaction, which is a well-known process for measuring cyanide in standard analytical methods. During the reaction, cyanide was converted to cyanogen chloride, which was extracted and collected on a paper impregnated with pyridine-barbituric acid. The color appeared on the paper was used to quantify the analyte using a scanning-assisted image processing software. The calibration curve was linear in the range of 3.0-100 g L - 1 with the detection limit of 0.7 µg L -1 . In the second study, a method combining headspace single-drop microextraction with a paper-based colorimetric assay was developed. Headspace single-drop microextraction using a microdrop containing unmodified gold nanoparticles (Au) as both the extractant and the colorimetric probe was used for determination of Se(IV). The method relied on the color change of the microdrop solution caused by the adsorption of in situ-generated hydrogen selenide on the surface of Au. Following extraction, the microdrop was spotted onto cellulose paper, and scanometric-assisted digital image analysis was used for quantification. The analytical variables affecting the method sensitivity were studied. The dynamic range was in the range of 15-100 ?g L -1 with the limit of quantification of 12 ?g L -1 . In the third study, a 96-well paper-based and laboratory setup-free optical platform was reported for the detection of butyrylcholinesterase enzyme (BChE) activity in human serum. Chromatographic paper was used to realize a novel analytical tool exploiting its porous structure for reagentless synthesize Prussian blue nanoparticles (, the sensing element), as well to load all the reagents required for the measurement. The principle of BChE activity detection relied on the reaction between the enzymatic by-product thiocholine and Prussian blue, giving the Prussian white with subsequently Prussian blue's fading, detected by a common office scanner supported by ImageJ software. Using this novel paper-based optical platform, BChE activity was linearly detected in the 2–15 U/mL range with a detection limit down to 0.8 U mL -1 . In the final study, a holistic use of paper-based substrate was described to detect copper ions in bodily fluid. Filter paper was highlighted to i) synthesize Au without the use of reductants and/or external stimuli, ii) print the electrodes, iii) pre-load the reagents for the assay, iv) filter the gross impurities, and v) pre-concentrate the target analyte, i.e. copper. With the regard to the last point, it represented the first application of paper-based substrate to pre-concentrate metals and improving the sensitivity of stripping analysis. Without the use of reagents/treatments by the end users, copper ions were detected down to 3 ?g L -1 , linearly from 10 to 400 ?g L -1 . The developed platform was applied towards the detection of copper ions in un-diluted sweat samples.