During the last decades the development of fluorescence techniques and their applications in bioscience and biotechnology has been accentuated. Due to high sensitivity, such techniques have become important tools in many research areas such as medical diagnosis, genetic analysis, structural mapping of cells, drug investigation, characterization of drug-metal complexes, analysis of drugs interaction with proteins, lipids and DNA. The local anesthetics are drugs that reversibly block the nervous impulse when applied to a limited region of the body. However, they possess a variety of additional effects in the biological systems. The fluoroquinolone derived antibiotics are synthetic antimicrobial agents that have been used clinically for more than 30 years. In addition to their antibacterial activity, some fluoroquinolones have been applied in the development of anticancer drugs and anti-HIV. The local pH of many biological environments has great influence in the spectroscopic properties of many drugs, including local anesthetics and fluoroquinolones. The ionization constants of the drugs, pKa, can be experimentally obtained via spectroscopic parameters such as absorbance, fluorescence intensity, and time-resolved fluorescence parameters. In this work we studied the local anesthetics dibucaine and tetracaine using steady state and time resolved fluorescence techniques. We also studied the antibiotics norfloxacin and levofloxacin and their complexes with gold. We determined the fluorescence lifetimes and described how the spectral fluorescence properties alter as a function of the pH. In the studies of fluorescence decay, we developed expressions for the pre-exponential factors and the fractional fluorescence intensities as a function of the pH for a fluorophore undergoing an acid-base transition. We showed that the pKa values are apparently shifted and obtained the expressions for the pK shifts. We determined the ionization constants, pKa from steady state fluorescence and from parameters associated with the lifetimes, and we tested the theoretical expressions using the experimental results. The observed pK shifts for dibucaine, levofloxacin and norfloxacin were found to agree with the theoretical expressions. The action of local anesthetics is believed to involve the interaction of the anesthetic molecules with lipids and/or membrane proteins. An analysis of this interaction is necessary to examine the anesthetic diffusion in the membrane and its effect in the organization and dynamics of the membrane lipids and proteins. We investigated the interaction of dibucaine with Na,K-ATPase enriched membranes, and the incorporation of the drug in the membrane. We determined the binding constants. The obtained values were 0.91 × 10(3) M(-1) for dibucaine and 0.77 × 10(3) M(-1) for tetracaine. We also studied the accessibility of the tryptophan residues using the fluorescence quenching by dibucaine. We obtained a dynamic quenching constant of 4.3 × 10(4) M(-1).