Quantification assays are often hampered by reagents used to prepare protein samples. Many have utilised the fluorescent properties of tryptophan to determine total protein and peptide content in order to circumvent these problems. Using Bovine serum albumin and TYK-nu cell lysates as standards, it was observed that tryptophan fluorescence readings for these purposes are only valid when the samples are sufficiently denatured. With this knowledge, we then conducted an investigation into where sample losses occur during the preparation of samples for proteomic experiments. In-solution digestion of the standards revealed sample dilution prior to the enzymatic digestion step was responsible for losses of up to half of the sample. However, protein digestion mitigated those losses to varying extents, largely because of the generation of peptides. The impact of different surfactants and reducing agents in establishing denaturing conditions for accurate protein or peptide quantification by tryptophan fluorescence will also be discussed.