Standardized evaluation of sperm quality is essential for research, commercial-scale cryopreservation, and induced spawning. However, standardized methods for evaluation of sperm bundles (spermatozeugmata or spermatophores) have not been established. The purpose of the present study was to use Redtail Splitfin (Xenotoca eiseni) as a model for freshwater livebearing fishes to establish initial standardized methods to collect sperm bundles, and quantitatively and qualitatively evaluate quality-related attributes. No sperm or sperm bundles were able to be collected by stripping. Testes were removed, rinsed, weighed, placed in 50 μL of buffer solution on a glass slide, and crushed gently 3-5 times with angled spade-tip forceps. Sperm bundles were released into the buffer solution and collected with a pipette into 1.5-mL centrifuge tubes. To quantify size and shape, images of bundles were captured with a CCD camera connected to a microscope, and measured with computer software. There was no significant correlation between body wet weight and major bundle axis length (P = 0.6759), minor axis length (P = 0.5658), average axis length (P = 0.5869), aspect ratio (P = 0.7839), and observed area (P = 0.5727). The concentrations of sperm bundles, estimated with the three methods (Makler® counting chamber, a hemocytometer, and direct counting) were significantly different (P < 0.0001). Hemocytometers were suitable for estimation of bundles from X. eiseni. To evaluate activation of sperm, bundles were viewed with a microscope, and classified into one of five phases by evaluating morphology of the bundles and motion of sperm within the bundles as Phase 0 through Phase 4 that represented early through late activation stages. The frequencies and duration of each activation phase were used to evaluate dissociation of sperm bundles and motility capability of sperm within the bundles. Within 180 min of activation, all five phases were observed. Overall, this study for the first time established standardized methods to collect and evaluate quality-related attributes of sperm bundles. These standardized evaluations provide a basis for further modification, standardization, and generalization, which are useful in research on livebearing fishes involving male gametes, such as studies on cryopreservation, artificial insemination, and in development of germplasm repositories for imperiled species including goodeids.