Despite > 60 years of conservation in Uganda's national parks the populations of lions and spotted hyaenas in these areas have never been estimated using a census method. Estimates for some sites have been extrapolated to other protected areas and educated guesses have been made but there has been nothing more definitive. We used a lure count analysis method of call-up counts to estimate populations of the lion Panthera leo and spotted hyaena Crocuta crocuta in the parks where reasonable numbers of these species exist: Queen Elizabeth Protected Area, Murchison Falls Conservation Area and Kidepo Valley National Park. We estimated a total of 408 lions and 324 hyaenas for these three conservation areas. It is unlikely that other conservation areas in Uganda host > 10 lions or > 40 hyaenas. The Queen Elizabeth Protected Area had the largest populations of lions and hyaenas: 140 and 211, respectively. It is estimated that lion numbers have declined by 30% in this protected area since the late 1990s and there are increasing concerns for the long-term viability of both species in Uganda.