The chiral nature of living systems has obvious implications for the biologically active compounds that interact with them. At the molecular level, chirality represents an intrinsic property of the essential building blocks of life, such as amino acids and sugars, and therefore, of peptides, proteins, enzymes, carbohydrates, nucleosides and a number of alkaloids and hormones. As a consequence, processes mediated by biological systems are stereochemistry-sensitive, and a pair of enantiomers can have different effects on living organisms. The scientific community has been studying the implications of chirality for life for more than a century. Today, it is still a topic of active research and debate due to the large number of chiral molecules that are part of living organisms and of our everyday life. In this context, analytical methods for the separation of the enantiomers of chiral molecules play a crucial role. Undoubtedly, the use of chiral stationary phases (CSPs) in high performance liquid chromatography (HPLC) is the preferred choice for enantioseparations. This is evidenced by the huge number of CSPs available on the market. This fact, together with the trial-and-error methods commonly used to select the most suitable chromatographic system (CSP/mobile phase combination) for a given enantioseparation, results in enormous cost and experimental effort. This makes it necessary to develop strategies to simplify this important task. This Doctoral Thesis has two clearly differentiated main objectives: (i) To contribute to the knowledge of chiral HPLC with polysaccharide-based CSPs (the most popular commercial ones: three amylose and five cellulose derivatives), and hydro-organic mobile phases (comprising acetonitrile (ACN) and methanol (MeOH) aqueous solutions compatible with aqueous matrices and mass spectrometry (MS) detection). To this end, the following specific objectives were set: (a) to contribute to a rational selection of the chromatographic system to separate the enantiomers of a given compound. To this end, the retention and enantioresolution of a large dataset of structurally unrelated chiral compounds (approximately 60 basic and neutral drugs and pesticides) in the chromatographic systems above-indicated is compared. Moreover, quantitative structure-property relationships (QSPRs) for enantioresolution related data obtained in some of the chromatographic systems studied are developed. (b) To explore the use of deconvolution of overlapping peaks to achieve the mathematical resolution when the baseline resolution cannot be achieved experimentally. To illustrate the potential of this peak model strategy, the enantioseparation of eight chiral drugs in five polysaccharide-based CSPs and ACN or MeOH hydro-organic mobile phases at different separation temperatures is considered. (ii) To contribute to the advancement of knowledge of the risks and hazards of chiral pollutants. To this end, OECD (Organisation for Economic Co-operation and Development) biodegradability tests using activated sludge from a Valencian waste water treatment plant (Quart Benàger) are performed for some common chiral pharmaceutical pollutants: trimeprazine, ibuprofen, ketoprofen, bupivacaine, mepivacaine, prilocaine and propanocaine. Next, the separation and determination of the enantiomers of the intact compound is performed using chiral HPLC methods (with amylose- or cellulose-based CSPs and ACN or MeOH hydro-organic mobile phases compatible with aqueous matrices and MS detection) developed for that purpose.