Defective interfering particles (DIPs) are virus mutants that lack essential genes for growth, but able to divert the viral proteins produced by an infectious virus, and thus, interfere with infectious virus production. The accumulating evidence about their presence in nature and the increasing interest in DIP-based vaccine formulations highlight the need for an accurate understanding of the effect of DIPs on virus growth at intracellular and cell-to-cell spread scale. To analyze the multi-scale effects of DIPs, we first tracked the intracellular growth of a recombinant vesicular stomatitis virus (VSV) in individual cells using fluorescence microscopy and quantified the effects of DIPs on intracellular virus production kinetics. Through a metric scoring the dose-dependent effects of DIPs in cells and their cell-to-cell variations, we identified two-fold higher degree of interference by DIPs in isolated cells than that in single cells within a population. Quantification of infection spread behavior revealed an induction in the diversity of spread patterns with increasing DIP input. Through a cellular automata model driven by the measured virus growth kinetics in individual cells, we found that the intracellular scale effects of DIPs and cell-to-cell variations play a major role in the formation of the diverse interfered spread patterns. Using the developed framework the DIP-induced interference was compared to the interference by a recombinant defective viral mutant that only lacks virus assembly proteins. Our findings suggested that during the DIP co-infections the competition at replication stage interferes mainly with the early growth delaying and reducing the intracellular virus activity, and thus decreasing extracellular virus production, which is further diminished by the competition at virus assembly stage. Despite this reduction in virus production, we observed that the enrichment of DIPs are limited by the growth of infectious virus suggesting the dependency of DIPs on infectious virus at all scales of virus infections. Overall, the insight provided by this work can guide future studies of the viral infectious disease progression in the presence of DIPs, while the developed framework sets a platform to investigate other microbial infections under the effects of different interfering substances, such as antiviral or antibacterial drugs and vaccines.