ABSTRACT The loss of virus particles is typically considered to arise from a first-order kinetic process. Signals of deviations from this exponential decay are often de-prioritized. Here, we propose methods to evaluate if a design is adequate to evaluate evidence for multiphasic virus particle decay and to optimize the sampling times of decay experiments, accounting for uncertainties in viral kinetics. First, we evaluate 1500 synthetic scenarios of biphasic decays, with varying decay rates and initial proportions of subpopulations. Robust inference of multiphasic decay is more likely when the faster decaying subpopulation predominates insofar as early samples are taken to resolve the faster decay rate. Overall, we find that design optimization leads to a better precision of estimation while reducing the number of samples. It helps to estimate adequately the fastest decay in 54% of situations vs. 41% using a non-optimized design. We then apply these methods to infer multiple decay rates associated with the decay of ΦD9, an evolved isolate derived from phage Φ21. A pilot experiment confirmed that ΦD9 decay is multiphasic, but was unable to resolve the rate or proportion of the fast decay subpopulation(s). We then applied optimal design methods to propose new ΦD9 sampling times. Using this strategy, we were able to robustly estimate both decay rates and their respective subpopulations. Notably, we conclude that the vast majority (94%) of the population decays at a rate 16-fold higher than a slow decaying population. Altogether, these results provide methods to quantitatively estimate heterogeneity in viral decay.