© 2019 Blackwell Verlag GmbH Spermatogonial stem cell (SSC) is known for its self-renewal capacity. We have studied the in vitro proliferation of isolated SSC from adult alpaca (Vicugna pacos) testes. A total of 107 samples were evaluated of which 31 were evaluated at baseline, 36 were cultivated in DMEM and 40 in STEMPRO media. Half of the cultivated samples was analysed after 14 days, and the rest after 21 days. Round cell subpopulations were identified with FITC-DBA by flow cytometry: strongly positive DBA (sDBA+) as SSC, weakly positive DBA (wDBA+) as in early differentiation and negative DBA as differentiated. At the beginning, 4.16% of the cells were SSC, 37.61% wDBA+ while 54.12% were DBA−. After 14 days, 42.28% of SSC, 44.68% wDBA+ and 11.07% DBA− were found in DMEM while 47.09% of SSC, 32.57% wDBA+ and 18.48% DBA− in STEMPRO. After 21 days 38.66% were SSC, 52.78% wDBA and 7.65% DBA− in DMEM and on STEMPRO media 47.92% SSC, 44.20% wDBA+, 4.93% DBA−. There is a significant difference between the number of initial and SSC cultivated, as well as between DBA− (p < 0.05), while there is no significant difference between the wDBA+ (p > 0.05). Our results suggest that both culture media are appropriate for the in vitro proliferation of alpacas SSC.