Methods and systems for determining a drop delay of a flow stream in a flow cytometer are provided. Aspects of the methods according to certain embodiments include capturing an image of the flow stream to obtain an imaged flow stream, identifying a disturbance at a break off point in the imaged flow stream and calculating the drop delay of the flow stream based on the identified disturbance. Systems for practicing the subject methods having an imaging sensor for capturing one or more images of the flow stream and a processor configured to identify a disturbance at a break off in the imaged flow stream and calculating the drop delay using the imaged flow stream are also provided. Non-transitory computer readable storage mediums are also described.