<p>Adeno-associated virus (AAV) vectors are made up of a protein shell that typically encapsidates a single stranded viral deoxyribonucleic acid (DNA) genome up to a total of ~ 4700 nucleotides. Classical AAV Manufacturing processes yield a mixture of capsids that either contain no payload, partial DNA, or the gene of interest (GOI). Several studies have generated variable data indicating that empty capsids (1) affect transduction efficiency by engaging the immune system’s neutralizing antibodies, (2) do not improve full capsid uptake, (3) cause liver toxicity at high capsid loads, or (4) alter biodistribution. To study the effect of different empty capsid ratios on transduction efficiency, multiple AAV serotypes and full-to-empty methods were utilized in conjunction with a 5-day<i> in vitro</i> potency method. Total protein expression levels for each AAV were measured by a luciferase reporter after differentiation of C2C12 mouse myoblast cells into myotubes. Empty capsid load regardless of serotype identity did yield a statistically significant change in total protein expression levels. This data suggests that limiting the number of empty capsids regardless of the serotype is advised to ensure the highest dose of GOI containing AAVs reach target cells. Manufacturing practices should continue to reduce the overall empty capsid load in each batch to ensure most AAV particles contain the GOI. The findings of this project could aid in process optimization and method development strategies for cell-based assays to quantify the level of protein expression in AAVs containing other GOIs.</p> Graphical Abstract <p></p>