The following are the International Society of Urological Pathology (ISUP) recommendations for the use of immunohistochemistry (IHC) in prostate specimens. Either high–molecular weight cytokeratin (34βE12 or CK5/6 or others) or p63 or a combination of the 2 with AMACR either in a double or triple cocktail is recommended for the workup of small foci of atypical glands suspicious for adenocarcinoma of the prostate (PCa). ERG is optional as it is present in only 40% to 50% of prostate cancers and also positive in high-grade prostatic intraepithelial neoplasia. In the setting of obvious carcinoma or benign glands, there is no justification to do basal cell stains and AMACR. If there is a Gleason score of 3+4=7 or a higher-grade cancer on at least 1 part, the workup of other parts with an atypical focus suspicious for Gleason score 3+3=6 cancer is not recommended. In the setting of Gleason score 4+3 or 4+4=8 cancer on at least 1 part, the extent of high-grade cancer could affect clinical treatment such that workup of other atypical possible high-grade cancer foci is justified. In the setting of Gleason score 4+3 or higher-grade cancer on at least 1 part, given that intraductal carcinoma in the vast majority of cases is considered extension of high-grade cancer into prostatic ducts and acini, it is not recommended in the setting of definitive invasive high-grade cancer that workup of additional cribriform lesions be pursued. In the setting of Gleason score 3+3 on at least 1 part, the number of positive cores and/or their location could possibly affect subsequent therapy in terms of suitability for active surveillance or focal therapy, such that unless one knows with certainty that it would not affect therapy, it is justified to perform an IHC workup of additional atypical foci. In the differential diagnosis of high-grade PCa versus urothelial carcinoma (UC), the primary option is to use prostate-specific antigen (PSA) as a first test to identify PCa and GATA3 to identify UC. If GATA3 is not available, then HMWCK and p63 can be used. If the tumor is PSA positive with intense staining and HMWCK and p63 negative, the findings are diagnostic of PCa. If the tumor is equivocal/weak/negative for PSA and negative/focal for p63 and HMWCK, then one needs to perform staining for P501S, NKX3.1, and GATA3. Some experts also include PAP in this second round of staining. If the tumor is negative for PSA and diffusely strongly positive for p63 and HMWCK, the findings are diagnostic of UC. If the tumor is negative for PSA and moderately to strongly positive for GATA3, it is diagnostic of UC. Laboratories should be encouraged to use GATA3 for UC and add P501S and NKX3.1 as prostate markers in addition to PSA, p63, and HMWCK. If GATA3, p501S, and NKX3.1 are not available in equivocal cases, the case should be sent out for consultation to laboratories with these antibodies. The article also covers the use of IHC in: (1) high-grade PCa versus bladder adenocarcinoma; (2) prostatic small cell carcinoma versus high-grade PCa; (3) metastatic carcinoma of unknown primary: rule out PCa; (4) nonspecific granulomatous prostatitis/xanthoma versus high-grade PCa; (5) adult prostate sarcoma versus sarcomatoid PCa; (6) colorectal adenocarcinoma versus high-grade PCa; and (7) prognostic IHC markers.