Foot-and-mouth disease (FMD) is a highly infectious disease of cattle and other cloven-hoofed animals, causing huge economic losses annually worldwide. This disease is endemic in Pakistan where the serotypes of the foot-and-mouth disease virus (FMDV) are A, O and ASIA-1. At present, trivalent FMDV vaccines are being used to prevent FMD but the current production process is laborious and is unable to fulfill the needs of the meat and dairy industries. To meet the vaccine needs of Pakistan, the conventional method of using adherent cell lines to produce the vaccine could be replaced by suspension cell cultures which produce higher yields in less time and less volume. Therefore, the aim of this study was to investigate and optimize some of the factors that affect viable cell density and subsequent virus yield. The relationship between the yield of the 146S fraction and the TCID50 of the virus preparations obtained was also evaluated as a mean to control and check the quality of the vaccine product. The results provided optimized conditions for vaccine production using cell suspensions and showed that there was a linear relationship between TCID50 and 146S fraction yield. Either TCID50 or the 146S fraction yield, or both could be used as parameters for quality monitoring during vaccine production. Using TCID50 reduced the number of steps involved in virus production while measuring 146S fraction yield was useful for quality control. However, more studies are required to evaluate the relative effectiveness of vaccines produced by virus cultures using either TCID50 or 146S fraction as quality monitoring tools.