Tenofovir (TFV; prescribed as the tenofovir disoproxil fumarate prodrug), an acyclic nucleotide analog, inhibits human immunodeficiency virus (HIV) replication and is currently used for both HIV treatment and prevention (as pre‐exposure prophylaxis). The pharmacologically active form of TFV is TFV‐diphosphate (TFV‐DP). Previously, we reported the heterogeneous distribution of TFV and TFV‐DP in colorectal tissue (a putative site of HIV infection) sections collected from research participants receiving a TFV‐containing enema. Although the observed heterogeneity in TFV‐DP distribution in colorectal tissue is multifactorial, it could be envisioned that dephosphorylation plays a role in regulating TFV‐DP levels since this molecule is structurally similar to ATP, which is a substrate for nucleotidases such as nucleoside triphosphate diphosphohydrolases (NTPDases). Therefore, in order to gain increased understanding of TFV‐DP disposition, the ability of recombinant human NTPDase 1, 3, 4, 5, and 8 to dephosphorylate TFV‐DP was investigated by incubating these enzymes with TFV‐DP, individually. TFV‐DP levels were measured using an ultra‐high‐performance liquid chromatography tandem mass spectrometry approach. From these incubations, TFV‐DP substrate depletion, a measure of dephosphorylation, was observed in the presence of NTPDase 1 or 3 enzymes. The activities of NTPDase 1 and 3 towards TFV‐DP were further confirmed using a malachite green phosphate assay that measures the release of inorganic phosphate. Further, using immunoblotting, we tested whether NTPDase 1 and 3 are expressed in colorectal tissue. These studies revealed the presence of both proteins in colorectal tissue lysate. In order to gain an understanding of the spatial distribution patterns of NTPDase 1 and 3 in colorectal tissue, Matrix‐Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) was employed. As a first step, we developed a MALDI MSI method to directly detect and identify peptides of NTPDase 1 and 3 following trypsin digestion. Trypsin digestions on NTPDases were carried out using a concentration of 0.05 μg/μl, and α‐cyano‐4‐hydroxycinnamic acid was used as the MALDI matrix. Following trypsin digestions and MALDI MS analysis, peptides corresponding to NTPDase 1 and 3 were detected and identified. From these, NTPDase 1 exhibited peptides, SLSNYPFDFQGAR, SQHQETPVYLGATAGMR, and IITGQEEGAYGWITINYLLGK whereas NTPDase 3 yielded following peptides: GSGISSYGNNPQDVPR, YGIVLDAGSSR, FTEETWPQIHFEK, and MFTVLTR. Thus, in this study, we established a MALDI MS methodology for detection and identification of peptides corresponding to NTPDase 1 and 3. Furthermore, the above developed method can be used to generate spatial distribution profiles (including cell‐type and region‐specific localization patterns) of NTPDase 1 and 3 in colorectal and other tissues of interest that are susceptible to HIV infection. Data from in vitro incubations indicate that NTPDase 1 and 3 may contribute to the regulation of TFV‐DP levels.Support or Funding InformationThis work was funded by NIH grants U19AI113127, UM1 AI068613 and R01AI128781.