This constitutes the first study to report on the use of a novel approach to determine inactivation in PUV-irradiated Cryptosporidium parvum oocysts suspended in water based on the measurement of cellular adenosine triphosphate (ATP) concentration. This study also compares the efficiency of a novel ATP assay to that of using the combined in vitro HCT-8 cell culture – quantitative polymerase chain reaction (qPCR) method for determining the inactivation in the waterborne pathogen C. parvum after exposure to pulsed UV (PUV) treatments. Findings were compared with using the combined cell culture-qPCR approach for determining oocyst viability in similarly treated samples. PUV effectively killed C. parvum with a 5.4 log10 loss in oocyst viability after exposure to a UV dose of 8.5 μJ/cm2 as determined by the in vitro cell culture – qPCR assay. The ATP assay was shown to be significantly less effective in measuring loss of oocyst viability in similarly PUV-irradiated samples for all combination of treatment regimes studied. Measurement of cellular ATP is not suitable as an indicator of the disinfection efficiency of PUV-irradiated C. parvum oocysts. The levels of ATP present post PUV-irradiated samples suggests that significant cellular activity remained in treated oocysts that are unable to invade human HCT-8 cells. However, further studies are merited to investigate factors that might aid repair post PUV treatments in this water-borne human parasite. Use of this ATP assay offers an interesting insight into loss of infectivity in PUV-treated C. parvum. This rapid assay does not appear suitable for investigating or optimizing treatment efficiency under varying operational settings as it detects PUV-treated oocysts at levels significantly higher compared with using the in vitro cell culture-qPCR infectivity assay. Overestimation of survivors by the ATP assay may suggest that a sub-population of C. parvum oocysts may exist in a viable but non-infectious state or may require a period of resuscitation to facilitate photo-repair (if possible) that may lead to regained ability to infective human hosts.