Flow virometry can support advanced water treatment and reuse by delivering near real-time information about viral water quality. Realizing the full potential of this technique, requires rigorous sample-preparation protocols as well as objective, automated methods for expediting and improving FVM data analysis. Below, we present an optimized sample-preparation protocol for FVM-based detection and enumeration of T4 bacteriophage, an environmentally relevant viral surrogate. The protocol, which was rigorously optimized using a fractional factorial experimental design, blends and improves on existing protocols developed using a traditional “pipeline”-style optimization approach. As use of FVM for viral water-quality assessment expands, we recommend that this protocol be used to validate instrument performance prior to and alongside application of FVM on environmental samples. Widespread adoption of a consistent, optimized analytical approach will engender confidence in FVM data and will facilitate cross-laboratory comparisons.