UV254 and SUVA254 determinations are used in water treatment to obtain information about the nature of dissolved organic carbon (DOC) in water samples. This article presents new information about obtaining accurate results for UV254 and SUVA254 (the ratio of UV254 to the concentration of DOC in the water) in freshwater samples. Although the potential impact of colloidal materials on UV254 results is mentioned in standard and US Environmental Protection Agency methods, these methods provide no clear guidance for validating such measurements. The main objectives of this research were to examine the effects of colloidal and particulate matter in freshwaters on DOC and UV254 determinations and to develop some guidelines for filtering freshwater samples with widely varying turbidities. The experimental results showed that the water sample turbidity level and characteristics as well as the nature of the membrane filter pore structures and pore size distribution play important roles in UV254 and SUVA254 determinations. Measuring turbidity in the sample filtrate was a simple way to validate UV254 and SUVA254 results. The findings of this study will help the water treatment industry obtain more reliable UV254 and SUVA254 results and assist in developing improved experimental protocols.