Many assays have been developed for the detection of influenza virus which is an important respiratory pathogen. Development of these assays commonly involves the use of human clinical samples for validation of their performance. However, clinical samples can be difficult to obtain, deteriorate over time, and be inconsistent in composition. The goal of this study was to develop a simulated respiratory secretion (SRS) that could act as a surrogate for clinical samples. To this end, we determined the effects major respiratory secretion components (Na+, K+, Ca2+, cells, albumin IgG, IgM, and mucin) have on the performance of influenza assays including both nucleic acid amplification and rapid antigen assays. Minimal effects on the molecular assays were observed for all of the components tested, except for serum derived human IgG, which suppressed the signal of the rapid antigen assays. Using dot blots we were able to show anti-influenza nucleoprotein IgG antibodies are common in human respiratory samples. We composed a SRS that contained mid-point levels of human respiratory sample components and studied its effect compared to phosphate buffered saline and virus negative clinical sample matrix on the Veritor, Sofia, CDC RT-PCR, Simplexa, cobas Liat, and Alere i influenza assays. Our results demonstrated that a SRS can interact with a variety of test methods in a similar manner to clinical samples with a similar impact on test performance.