Aim of this study was to delineate sample handling procedures for accurate fluorescence and UV absorbance measurements of wastewater organic matter. Investigations were performed using different wastewater qualities, including primary, secondary and tertiary wastewater effluents, and a wastewater-impacted surface water. Filtration by 0.7 μm glass microfiber filter, 0.45 μm polyvinylidene fluoride (PVDF) membrane, 0.45 μm cellulose nitrate membrane, and 0.45 μm polyethersulfone (PES) syringe filter released manufacture impurities in water that affected fluorescence measurements. However, pre-washing of filter by Milli-Q water was able to eliminate these interferences. Different storage conditions were tested, including storage of filtered and unfiltered samples under different temperatures (25 °C, 4 °C, -20 °C). According to the obtained results, the best practice of wastewater samples preservation was sample filtration at 0.7/0.45 μm immediately after collection followed by storage at 4 °C. However, the time of storage that assured changes of these spectroscopic measurements that do not exceed the 10% of the original value was dependent on water quality and selected wavelengths (i.e., selected fluorescing organic matter component). As a general rule, it is advisable to perform fluorescence and UV absorbance measurements as soon as possible after collection avoiding storage times of filtered water longer than 2 days. Finally, addition of chlorine doses typical for wastewater disinfection mainly affected tryptophan-like components, where changes that exceed the 10% of the fluorescence intensity measured in the unchlorinated sample were observed even at very low doses (≥1 mg/L). On the contrary, tyrosine-like and humic-like components showed changes <10% at chlorine doses of 0.5-5 mg/L.